Allogeneic and xenogeneic transplantation

ABSTRACT

The invention provides methods for restoring or inducing imnmunocompetence, the methods including the step of introducing donor thymic tissue into the recipient. The invention also provides methods for inducing tolerance in a recipient including introducing donor thymic tissue into the recipient. The invention further provides methods of inducing tolerance including administering to the recipient a short course of help reducing treatment or administering a short course and methods of prolonging the acceptance of a graft by administering a short course of an immunosuppressant.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 09/126,704, filed onJul. 30, 1998, which is a continuation of U.S. Ser. No. 08/458,720,filed on Jun. 1, 1995, now U.S. Pat. No. 5,876,708, which is acontinuation-in-part of: U.S. Ser. No. 08/266,427, filed on Jun. 27,1994 now U.S. Pat. No. 5,614,187; U.S. Ser. No. 08/451,210, filed on May26, 1995, which is a file wrapper continuation of U.S. Ser. No.07/838,595, filed on Feb. 19, 1992, now abandoned; U.S. Ser. No.08/220,371, filed on Mar. 29, 1994, now abandoned; PCT/US94/05527 filedon May 16, 1994; U.S. Ser. No. 08/08/243,653, filed on May 16, 1994, nowU.S. Pat. No. 5,658,564; U.S. Ser. No. 08/114,072, filed on Aug. 30,1993, now U.S. Pat. No. 5,624,823; U.S. Ser. No. 08/150,739, filed onNov. 10, 1993, now abandoned; U.S. Ser. No. 08/212,228, filed on Mar.14, 1994, now abandoned; and PCT/US94/01616 filed on Feb. 14, 1994. Allof the above-referenced applications are hereby incorporated byreference.

BACKGROUND OF THE INVENTION

This invention relates to the replacement of thymus function and to theinduction or restoration of immunological tolerance. The inventionfurther relates to tissue and organ transplantation.

The thymus is the central organ for the development of mature,self-tolerant T cells that recognize peptide antigens in the context ofself major histocompatibility (MHC) antigens. The requirement for selfMHC molecules to present antigen is termed MHC restriction. Athymicindividuals do not have an organ in which to generate normal numbers ofMHC restricted T cells and are therefore immunoincompetent.

SUMMARY OF THE INVENTION

It has been discovered that host T cells of an athymic T cell depletedhost which has received a thymic graft, e.g., a xenogeneic thymic graft,can mature in the donor thymic tissue, e.g., in xenogeneic thymictissue. Host T cells which mature in the implanted xenogeneic thymictissue are immunocompetent.

Accordingly, the invention features, in one aspect, a method ofrestoring or inducing immunocompetence (or restoring or promoting thethymus-dependent ability for T cell progenitors to mature or developinto functional mature T cells) in a host or recipient, e.g., a primatehost or recipient, e.g., a human, which is capable of producing T cellprogenitors but which is thymus-function deficient and thus unable toproduce a sufficient number of mature functional T cells for a normalimmune response. The invention includes the step of introducing into theprimate host, donor thymic tissue, e.g., xenogeneic thymic tissue,preferably fetal or neonatal thymic tissue, so that host T cells canmature in the implanted thymic tissue.

In preferred embodiments the donor of the thymic tissue is a xenogeneicspecies and: the thymic xenograft is a discordant xenograft; the thymicxenograft is a concordant xenograft; the host is a primate, e.g., ahuman, and the thymic tissue is swine, e.g., miniature swine, thymictissue, or primate thymic tissue.

The method can include other steps which facilitate acceptance of thedonor tissue, or otherwise optimize the method. In preferred embodimentsthe thymic tissue is xenogeneic and: liver or spleen tissue, preferablyfetal or neonatal liver or spleen tissue, is implanted with the thymictissue; donor hematopoietic cells, e.g., cord blood stem cells or fetalor neonatal liver or spleen cells, are administered to the recipient,e.g., a suspension of fetal liver cells is administeredintraperitoneally or intravenously; the recipient is thymectomized,preferably before or at the time the xenograft thymic tissue isintroduced.

In other preferred embodiments the method includes: (preferably prior toor at the time of introducing the thymic tissue into the recipient)depleting, inactivating or inhibiting recipient natural killer (NK)cells, e.g., by introducing into the recipient an antibody capable ofbinding to NK cells of the recipient, to prevent NK mediated rejectionof the thymic tissue; (preferably prior to or at the time of introducingthe thymic tissue into the recipient) depleting, inactivating orinhibiting host T cell function, e.g., by introducing into the recipientan antibody capable of binding to T cells of the recipient; (preferablyprior to or at the time of introducing the thymic tissue into therecipient) depleting, inactivating or inhibiting host CD4⁺ cellfunction, e.g., by introducing into the recipient an antibody capable ofbinding to CD4, or CD4⁺ cells of the recipient.

Other preferred embodiments include the step of (preferably prior tothymic tissue or hematopoietic stem cell transplantation) creatinghematopoietic space, e.g., by one or more of: irradiating the recipientmammal with low dose, e.g., between about 100 and 400 rads, whole bodyirradiation, the administration of a myelosuppressive drug, or theadministration of a hematopoietic stem cell inactivating or depletingantibody, to deplete or partially deplete the bone marrow of therecipient (preferably prior to thymic tissue transplantation).

Other preferred embodiments include (preferably prior to thymic tissueor hematopoietic stem cell transplantation) inactivating thymic T cellsby one or more of: irradiating the host with, e.g., about 700 rads ofthymic irradiation, administering to the recipient one or more doses ofan anti T cell antibody, e.g., an anti-CD4 and/or an anti-CD8 monoclonalantibody, or administering to the recipient a short course of animmunosuppressant, as is described in U.S. Ser. No. 08/220,371 andfurther described below.

Other preferred embodiments include depleting or otherwise inactivatingnatural antibodies, e.g., by one or more of: the administration of adrug which depletes or inactivates natural antibodies, e.g.,deoxyspergualin; the administration of an anti-IgM antibody; or theadsorption of natural antibodies from the host's blood, e.g., bycontacting the host's blood with donor antigen, e.g., by hemoperfusionof a donor organ, e.g., a kidney or a liver, from the donor species.

In preferred embodiments the host or recipient is a post-natalindividual, e.g., an adult, or a child.

In preferred embodiments the method further includes the step ofidentifying a host or recipient which is capable of producing T cellprogenitors but which is thymus-function deficient and thus unable toproduce a sufficient number of mature functional T cells for a normalimmune response.

In other preferred embodiments, a graft which is obtained from adifferent organ than is the thymic tissue is implanted in the recipient;and the recipient does not receive hematopoietic stem cells, e.g., bonemarrow cells, from the donor or the donor species.

Other methods can be combined with the methods disclosed herein topromote the acceptance of the graft by the recipient. For example,tolerance to the donor tissue can also be induced by inserting a nucleicacid which expresses a donor antigen, e.g., a donor MHC gene, into acell of the recipient, e.g., a hematopoietic stem cell, and introducingthe genetically engineered cell into the recipient. For example, humanrecipient stem cells can be engineered to express a swine MHC gene,e.g., a swine class I or class II MHC gene, or both a class I and aclass II gene, and the cells implanted in a human recipient who willreceive swine thymic tissue. When inserted into a recipient primate,e.g., a human, expression of the donor MHC gene results in tolerance tosubsequent exposure to donor antigen, and can thus induce tolerance tothymic tissue from the donor. These methods, and other methods which canbe combined with the methods disclosed herein, are discussed in Sachs,U.S. Ser. No. 08/126, 122, filed Sep. 23, 1993, hereby incorporated byreference and in Sachs, U.S. Ser. No. 08/129,608, filed Sep. 29, 1993,hereby incorporated by reference.

Methods of inducing tolerance, e.g., by the implantation ofhematopoietic stem cells, disclosed in Sachs, Cosimi, and Sykes, U.S.Ser. No. 07/838,595, filed Feb. 19, 1992, hereby incorporated byreference, can also be combined with the methods disclosed herein.

Other methods of inducing tolerance may also be used to promoteacceptance of the donor tissue. For example, suppression of T cell help,which can be induced, e.g., by the administration of a short course ofhigh dose immunosuppressant, e.g., cyclosporine, has been found toinduce tolerance. In these methods, T cell help is suppressed for acomparatively short period just subsequent to implantation of a graft,and does not require or include chronic immunosuppression. Thesemethods, as well as other methods which can be combined with the methodsdisclosed herein, are described in Sachs, U.S. Ser. No. 08/220,371,filed Mar. 29, 1994, hereby incorporated by reference.

Other methods of promoting tolerance or promoting the acceptance ofgrafts, e.g., by altering levels of cytokine activity, are disclosed inSachs, LeGuern, Sykes, and Blancho, U.S. Ser. No. 08/114,072, filed Aug.30, 1993, hereby incorporated by reference.

It has also been discovered that xenogeneic thymic tissue can be used toinduce tolerance to a xenogeneic graft in a recipient.

Accordingly, in another aspect, the invention features, a method ofinducing tolerance in a recipient mammal, e.g., a primate, e.g., ahuman, of a first species to a graft obtained from a mammal of a secondspecies, e.g., a discordant species. The method includes: prior to orsimultaneous with transplantation of the graft, introducing into therecipient mammal thymic tissue, e.g., thymic epithelium, preferablyfetal or neonatal thymic tissue, of the second species; and (optionally)implanting the graft in the recipient. The thymic tissue prepares therecipient for the graft that follows, by inducing immunologicaltolerance at the T-cell level.

In preferred embodiments: the thymic xenograft is a discordantxenograft; the thymic xenograft is a concordant xenograft; the recipientis a human and the thymic tissue is swine, e.g., miniature swine, thymictissue, or primate thymic tissue.

Preferred embodiments include other steps to promote acceptance of thegraft thymus and the induction of immunological tolerance or tootherwise optimize the procedure. In preferred embodiments: liver orspleen tissue, preferably fetal or neonatal liver or spleen tissue, isimplanted with the thymic tissue; donor hematopoietic cells, e.g., cordblood stem cells or fetal or neonatal liver or spleen cells, areadministered to the recipient, e.g., a suspension of fetal liver cellsadministered intraperitoneally or intravenously; the recipient isthymectomized, preferably before or at the time the xenograft thymictissue is introduced.

In other preferred embodiments the method includes (preferably prior toor at the time of introducing the thymic tissue or stem cells into therecipient) depleting, inactivating or inhibiting recipient NK cells,e.g., by introducing into the recipient an antibody capable of bindingto natural killer (NK) cells of the recipient, to prevent NK mediatedrejection of the thymic tissue; (preferably prior to or at the time ofintroducing the thymic tissue into the recipient) depleting,inactivating or inhibiting recipient T cells, e.g., by introducing intothe recipient an antibody capable of binding to T cells of therecipient; (preferably prior to or at the time of introducing the thymictissue or stem cells into the recipient) depleting, inactivating orinhibiting host CD4⁺ cell function, e.g., by introducing into therecipient an antibody capable of binding to CD4, or CD4⁺ cells of therecipient. An anti-mature T cell antibody which lyses T cells as well asNK cells can be administered. Lysing T cells is advantageous for boththymic tissue and xenograft survival. Anti-T cell antibodies arepresent, along with anti-NK antibodies, in anti-thymocyte anti-serum.Repeated doses of anti-NK or anti-T cell antibody may be preferable.Monoclonal preparations can be used in the methods of the invention.

Other preferred embodiments include those in which: the recipient doesnot receive hematopoietic cells from the donor or the donor species: thesame mammal of the second species is the donor of both the graft and thethymic tissue; the donor mammal is a swine, e.g., a miniature swine; ananti-human thymocyte polyclonal anti-serum, obtained, e.g., from a horseor pig is administered to the recipient.

Other preferred embodiments include the step of (preferably prior tothymic tissue or hematopoietic stem cell transplantation) creatinghematopoietic space, e.g., by one or more of: irradiating the recipientmammal with low dose, e.g., between about 100 and 400 rads, whole bodyirradiation, the administration of a myelosuppressive drug, theadministration of a hematopoietic stem cell inactivating or depletingantibody, to deplete or partially deplete the bone marrow of therecipient.

Other preferred embodiments include (preferably prior to thymic tissueor hematopoietic stem cell transplantation) inactivating thymic T cellsby one or more of: irradiating the recipient with, e.g., about 700 radsof thymic irradiation, administering to the recipient one or more dosesof an anti T cell antibody, e.g., an anti-CD4 and/or an anti-CD8monoclonal antibody, or administering to the recipient a short course ofan immunosuppressant, as is described in U.S. Ser. No. 08/220,371 andfurther described below.

In preferred embodiments the host or recipient is a post-natalindividual, e.g., an adult, or a child.

In preferred embodiments the method further includes the step ofidentifying a host or recipient which is in need of a graft.

Other preferred embodiments include depleting or otherwise inactivatingnatural antibodies, e.g., by one or more of: the administration of adrug which depletes or inactivates natural antibodies, e.g.,deoxyspergualin; the administration of an anti-IgM antibody; or theadsorption of natural antibodies from the recipient's blood, e.g., bycontacting the hosts blood with donor antigen, e.g., by hemoperfusion ofa donor organ, e.g., a kidney or a liver, from the donor species.

Other methods can be combined with the methods disclosed herein topromote the acceptance of the graft by the recipient. For example,tolerance to the xenogeneic thymic tissue can also be induced byinserting a nucleic acid which expresses a donor antigen, e.g., a donorMHC gene, into a cell of the recipient, e.g., a hematopoietic stem cell,and introducing the genetically engineered cell into the recipient. Forexample, human recipient stem cells can be engineered to express a swineMHC gene, e.g., a swine class I or class II MHC gene, or both a class Iand class II gene, and the cells implanted in a human recipient who willreceive swine thymic tissue. When inserted into a recipient primate,e.g., a human, expression of the donor MHC gene results in tolerance tosubsequent exposure to donor antigen, and can thus induce tolerance tothymic tissue from the donor. These methods, and other methods which canbe combined with the methods disclosed herein, are discussed in Sachs,U.S. Ser. No. 08/126,122, filed Sep. 23, 1993, and in Sachs, U.S. Ser.No. 08/129,608, filed Sep. 29, 1993.

Methods of inducing tolerance, e.g., by the implantation ofhematopoietic stem cells, disclosed in Sachs, Cosimi, and Sykes, U.S.Ser. No. 07/838,595, filed Feb. 19, 1992, can also be combined with themethods disclosed herein.

Other methods of inducing tolerance may also be used to promoteacceptance of the xenogeneic thymic tissue. For example, suppression ofT cell help, which can be induced, e.g., by the administration of ashort course of high dose immunosuppressant, e.g., cyclosporine, hasbeen found to induce tolerance. In these methods, T cell help issuppressed for a comparatively short period just subsequent toimplantation of a graft, and does not require or include chronicimmunosuppression. These methods, as well as other methods which can becombined with the methods disclosed herein, are described in Sachs, U.S.Ser. No. 08/220,371, filed Mar. 29, 1994.

Other methods of promoting tolerance or promoting the acceptance ofgrafts, e.g., by altering levels of cytokine activity, are disclosed inSachs, LeGuern, Sykes, and Blancho, U.S. Ser. No. 08/114,072 filed Aug.30, 1993.

In another aspect, the invention features, a method of restoring orinducing immunocompetence in a recipient, e.g., a primate recipient,e.g., a human, at risk for an acquired immune disorder, (e.g., a humanat risk for AIDS), which is capable of producing T cell progenitors butwhich is thymus-function deficient and thus unable to produce asufficient number of mature functional T cells to provide a normalimmune response. The invention includes the steps of introducing intothe primate recipient, donor thymic tissue, e.g., xenogeneic thymictissue, so that recipient T cells can mature in the implanted donorthymic tissue. The thymic tissue is preferably fetal or neonatal thymictissue.

In preferred embodiments the thymic tissue is xenogeneic and: the thymicxenograft is a discordant xenograft; the thymic xenograft is aconcordant xenograft; the recipient is a human and the thymic tissue isvertebrate, e.g., swine, e.g., miniature swine, thymic tissue, orprimate thymic tissue.

Acceptance of a graft, especially a xenogeneic graft, will depend on thestage of the immune disorder. Generally, the more advanced the disorderthe more compromised the recipient immune system and the easier it is toinduce acceptance of donor thymic tissue. In some cases, the tolerizingeffect of the graft itself will be sufficient to provide for acceptanceof xenogeneic thymus. In other cases, additional measures will beneeded. Thus, the method can include other steps which facilitateacceptance of the donor tissue or otherwise optimize the method. Inpreferred embodiments: liver or spleen tissue, preferably fetal orneonatal liver or spleen tissue, is implanted with the thymic tissue;donor hematopoietic cells, e.g., cord blood stem cells or fetal orneonatal liver or spleen cells, are administered to the recipient, e.g.,a suspension of fetal liver cells is administered intraperitoneally orintravenously; the recipient is thymectomized, preferably before or atthe time the xenograft thymic tissue is introduced.

In preferred embodiments: the method includes (preferably prior to or atthe time of introducing the thymic tissue into the recipient) depleting,inactivating or inhibiting recipient NK cells, e.g., by introducing intothe recipient an antibody capable of binding to natural killer (NK)cells of the recipient, to prevent NK mediated rejection of the thymictissue; the method includes (preferably prior to or at the time ofintroducing the thymic tissue into the recipient), depleting,inactivating or inhibiting recipient T cells, e.g., by introducing intothe recipient an antibody capable of binding to T cells of therecipient; (preferably prior to or at the time of introducing the thymictissue into the recipient) depleting, inactivating or inhibiting hostCD4⁺ cell function, e.g., by introducing into the recipient an antibodycapable of binding to CD4, or CD4⁺ cells of the recipient.

Other preferred embodiments include the step of (preferably prior tothymic tissue or hematopoietic stem cell transplantation) creatinghematopoietic space, e.g., by one or more of: irradiating the recipientmammal with low dose, e.g., between about 100 and 400 rads, whole bodyirradiation, the administration of a myelosuppressive drug, theadministration of a hematopoietic stem cell inactivating or depletingantibody, to deplete or partially deplete the bone marrow of therecipient.

Other preferred embodiments include (preferably prior to thymic tissueor hematopoietic stem cell transplantation) inactivating thymic T cellsby one or more of: irradiating the recipient mammal with, e.g., about700 rads of thymic irradiation, administering to the recipient one ormore doses of an anti T cell antibody, e.g., an anti-CD4 and/or ananti-CD8 monoclonal antibody, or administering to the recipient a shortcourse of an immunosuppressant, as is described in U.S. Ser. No.08/220,371 and further described below.

Other preferred embodiments include depleting or otherwise inactivatingnatural antibodies, e.g., by one or more of: the administration of adrug which depletes or inactivates natural antibodies, e.g.,deoxyspergualin; the administration of an anti-IgM antibody; or theadsorption of natural antibodies from the recipient's blood, e.g., bycontacting the hosts blood with donor antigen, e.g., by hemoperfusion ofa donor organ, e.g., a kidney or a liver, from the donor species.

In preferred embodiments the host or recipient is a post-natalindividual, e.g., an adult, or a child.

In preferred embodiments the method further includes the step ofidentifying a host or recipient which is at risk for an acquired immunedisorder, (e.g., a human at risk for AIDS), which is capable ofproducing T cell progenitors but which is thymus-function deficient andthus unable to produce a sufficient number of mature functional T cellsto provide a normal immune response.

Other methods can be combined with the methods disclosed herein topromote the acceptance of the thymic graft by the recipient. Forexample, tolerance to donor tissue can also be induced by inserting anucleic acid which expresses a donor antigen, e.g., a donor MHC gene,into a cell of the recipient, e.g., a hematopoietic stem cell, andintroducing the genetically engineered cell into the recipient. Forexample, human recipient stem cells can be engineered to express a swineMHC gene, e.g., a swine class I or class II MHC gene, or both a class Iand a class II MHC gene, and the cells implanted in a human recipientwho will receive swine thymic tissue. When inserted into a recipientprimate, e.g., a human, expression of the donor MHC gene results intolerance to subsequent exposure to donor antigen, and can thus inducetolerance to thymic tissue from the donor. These methods, and othermethods which can be combined with the methods disclosed herein, arediscussed in Sachs, U.S. Ser. No. 08/126, 122, filed Sep. 23, 1993,hereby incorporated by reference and in Sachs, U.S. Ser. No. 08/220,371,filed Mar. 29, 1994.

Methods of inducing tolerance, e.g., by the implantation ofhematopoietic stem cells, disclosed in Sachs, Cosimi, and Sykes, U.S.Ser. No. 07/838,595, filed Feb. 19, 1992, can also be combined with themethods disclosed herein.

Other methods of inducing tolerance may also be used to promoteacceptance of the donor thymic tissue. For example, suppression of Tcell help, which can be induced, e.g., by the administration of a shortcourse of high dose immunosuppressant, e.g., cyclosporine, has beenfound to induce tolerance. In these methods, T cell help is suppressedfor a comparatively short period just subsequent to implantation of agraft, and does not require or include chronic immunosuppression. Thesemethods, as well as other methods which can be combined with the methodsdisclosed herein, are described in Sachs, U.S. Ser. No. 08/220,371,filed Mar. 29, 1994.

Other methods of promoting tolerance or promoting the acceptance ofgrafts, e.g., by altering levels of cytokine activity, are disclosed inSachs, LeGuern, Sykes, and Blancho, U.S. Ser. No. 08/114,072, filed Aug.30, 1993.

In another aspect, the invention features, a method of restoring orinducing immunocompetence in a recipient, e.g., a primate recipient,e.g., a human, at risk for an acquired immune disorder, (e.g., a humanat risk for AIDS) which is unable to produce a normal or sufficientnumber of mature functional T cells to provide normal immune function.The invention includes the steps of introducing into the primaterecipient, donor hematopoietic stem cells, so that donor T cells canmature in the recipient thymus.

In preferred embodiments the donor stem cells are from a xenogeneicdonor and: the xenograft hematopoietic stem cells are from a discordantspecies; the hematopoietic stem cells are from a concordant species; therecipient is a human and the hematopoietic stem cells are vertebrate,e.g., swine, e.g., miniature swine, hematopoietic stem cells, or primatehematopoietic stem cells.

Acceptance of the donor cells will depend on the stage of the immunedisorder. Generally, the more advanced the disorder the more compromisedthe recipient immune system and the easier it is to induce acceptance ofdonor, especially xenogeneic donor, tissue. In some cases, thetolerizing effect of the stem cells themselves will be sufficient toprovide for acceptance. In other cases, additional measures will beneeded. Thus, the method can include other steps which facilitateacceptance of the donor cells or otherwise optimize the method. Inpreferred embodiments: liver or spleen tissue, preferably fetal orneonatal liver or spleen tissue, is implanted with the donorhematopoietic cells, e.g., cord blood stem cells or fetal or neonatalliver or spleen cells, are administered to the recipient, e.g., asuspension of fetal liver cells is administered intraperitoneally orintravenously.

In preferred embodiments: the method includes, (preferably prior to orat the time of introducing the donor cells into the recipient)depleting, inactivating or inhibiting recipient NK cells, e.g., byintroducing into the recipient an antibody capable of binding to naturalkiller (NK) cells of the recipient, to prevent NK mediated rejection ofthe thymic tissue; the method includes (preferably prior to or at thetime of introducing the thymic tissue into the recipient), depleting,inactivating or inhibiting recipient T cells, e.g., by introducing intothe recipient an antibody capable of binding to T cells of therecipient; (preferably prior to or at the time of introducing the thymictissue into the recipient) depleting, inactivating or inhibiting hostCD4⁺ cell function, e.g., by introducing into the recipient an antibodycapable of binding to CD4, or CD4⁺ cells of the recipient.

Other preferred embodiments include the step of (preferably prior tothymic tissue or hematopoietic stem cell transplantation) creatinghematopoietic space, e.g., by one or more of: irradiating the recipientmammal with low dose, e.g., between about 100 and 400 rads, whole bodyirradiation, the administration of a myelosuppressive drug, theadministration of a hematopoietic stem cell inactivating or depletingantibody, to deplete or partially deplete the bone marrow of therecipient.

Other preferred embodiments include (preferably prior to thymic tissueor hematopoietic stem cell transplantation) inactivating thymic T cellsby one or more of: irradiating the recipient mamma i with, e.g., about700 rads of thymic irradiation, administering to the recipient one ormore doses of an anti T cell antibody, e.g., an anti-CD4 and/or ananti-CD8 monoclonal antibody, or administering to the recipient a shortcourse of an immunosuppressant, as is described in U.S. Ser. No.08/220,371 and further described below.

Other preferred embodiments include depleting or otherwise inactivatingnatural antibodies, e.g., by one or more of: the administration of adrug which depletes or inactivates natural antibodies, e.g.,deoxyspergualin; the administration of an anti-IgM antibody; or theadsorption of natural antibodies from the recipient's blood, e.g., bycontacting the hosts blood with donor antigen, e.g., by hemoperfusion ofa donor organ, e.g., a kidney or a liver, from the donor species.

In preferred embodiments the host or recipient is a post-natalindividual, e.g., an adult, or a child.

In preferred embodiments the method further includes the step ofidentifying a host or recipient which is at risk for an acquired immunedisorder, (e.g., a human at risk for AIDS) and which is unable toproduce a normal or sufficient number of mature functional T cells toprovide normal immune function.

Other methods can be combined with the methods disclosed herein topromote the acceptance of the transplanted stem cells by the recipient.For example, tolerance to donor tissue can be induced by inserting anucleic acid which expresses a donor antigen, e.g., a donor MHC gene,into a cell of the recipient, e.g., a hematopoietic stem cell, andintroducing the genetically engineered cell into the recipient. Forexample, human recipient stem cells can be engineered to express a swineMHC gene, e.g., a swine class I or class II MHC gene, or both a class Iand a class II gene, and the cells implanted in a human recipient whowill receive swine thymic tissue. When inserted into a recipientprimate, e.g., a human, expression of the donor MHC gene results intolerance to subsequent exposure to donor antigen, and can thus inducetolerance to tissue from the donor. These methods, and other methodswhich can be combined with the methods disclosed herein, are discussedin Sachs, U.S. Ser. No. 08/126, 122, filed Sep. 23, 1993, and in Sachs,U.S. Ser. No. 08/220,371, filed Mar. 29, 1994.

Methods of inducing tolerance, e.g., by the implantation ofhematopoietic stem cells, disclosed in Sachs, Cosimi and Sykes, U.S.Ser. No. 07/838,595, filed Feb. 19, 1992, can also be combined with themethods disclosed herein.

Other methods of inducing tolerance can be combined with the methodsdisclosed herein to promote acceptance of donor tissue. For example,suppression of T cell help, which can be induced, e.g., by theadministration of a short course of high dose immunosuppressant, e.g.,cyclosporine, has been found to induce tolerance. In these methods, Tcell help is suppressed for a comparatively short period just subsequentto implantation of a graft, and does not require or include chronicimmunosuppression. These methods, as well as other methods which can becombined with the methods disclosed herein, are described in Sachs, U.S.Ser. No. 08/220,371, filed Mar. 29, 1994.

Other methods of promoting tolerance or promoting the acceptance ofgrafts, e.g., by altering levels of cytokine activity, are disclosed inSachs, LeGuern, Sykes, and Blancho, U.S. Ser. No. 08/114,072, filed Aug.30, 1993.

In another aspect, the invention features, a method of restoring orinducing immunocompetence in a recipient, e.g., a primate recipient,e.g., a human, at risk for an acquired immune disorder, (e.g., a humanat risk for AIDS), which is thymus-function deficient and thus unable toproduce a normal number of mature functional T cells or a sufficientnumber of mature functional T cells for a normal immune response. Theinvention includes the steps of introducing into the primate recipient,donor thymic tissue, preferably, xenogeneic thymic tissue, and donorhematopoietic stem cells, preferably xenogeneic hematopoietic stemcells, so that donor T cells can mature in the implanted donor thymictissue. The thymic tissue is preferably fetal or neonatal thymic tissue.

In preferred embodiments the thymic graft is a xenograft and: the thymicxenograft is a discordant xenograft; the thymic xenograft is aconcordant xenograft; the recipient is a human and the thymic tissue isvertebrate, e.g., swine, e.g., immature swine, thymic tissue, or primatethymic tissue.

In preferred embodiments: the xenograft hematopoietic stem cells arefrom a discordant species; the hematopoietic stem cells are a concordantspecies; the recipient is a human and the hematopoietic stem cells arevertebrate, e.g., swine, e.g., miniature swine, hematopoietic stemcells, or primate hematopoietic stem cells.

In preferred embodiments the donor of the thymic graft and the donor ofthe stem cells are: the same organism; from the same species; syngeneic;matched at least one MHC locus; matched at least one class I MHC locus;matched at least one class II MHC locus; sufficiently MHC matched thatone will not reject a graft from the other; miniature swine from a herdwhich is completely or partially inbred.

Acceptance of donor tissue, especially xenogeneic tissue, will depend onthe stage of the immune disorder. Generally, the more advanced thedisorder the more compromised the recipient immune system and the easierit is to induce acceptance of donor tissue. In some cases, thetolerizing effect of the donor tissue itself will be sufficient toprovide for acceptance. In other cases, additional measures will beneeded. Thus, the method can include other steps which facilitateacceptance of donor tissue or otherwise optimize the method. Inpreferred embodiments: liver or spleen tissue, preferably fetal orneonatal liver or spleen tissue, is implanted with the thymic tissue;donor hematopoietic cells, e.g., cord blood cells or fetal or neonatalliver or spleen cells, are administered to the recipient, e.g., asuspension of fetal liver cells is administered intraperitoneally orintravenously; the recipient is thymectomized, preferably before or atthe time the xenograft thymic tissue is introduced.

In preferred embodiments: the method includes, (preferably prior to orat the time of introducing the thymic tissue or stem cells into therecipient) depleting, inactivating or inhibiting recipient NK cells,e.g., by introducing into the recipient an antibody capable of bindingto natural killer (NK) cells of the recipient, to prevent NK mediatedrejection of the thymic tissue; the method includes, (preferably priorto or at the time of introducing the thymic tissue or stem cells intothe recipient) depleting, inactivating or inhibiting recipient T cells,e.g., by introducing into the recipient an antibody capable of bindingto T cells of the recipient mammal; (preferably prior to or at the timeof introducing the thymic tissue or stem cells into the recipient)depleting, inactivating or inhibiting host CD4⁺ cell function, e.g., byintroducing into the recipient an antibody capable of binding to CD4, orCD4⁺ cells of the recipient.

Other preferred embodiments include the step of (preferably prior tothymic tissue or hematopoietic stem cell transplantation) creatinghematopoietic space, e.g., by one or more of: irradiating the recipientmammal with low dose, e.g., between about 100 and 400 rads, whole bodyirradiation, the administration of a myelosuppressive drug, theadministration of a hematopoietic stem cell inactivating or depletingantibody, to deplete or partially deplete the bone marrow of therecipient.

Other preferred embodiments include (preferably prior to thymic tissueor hematopoietic stem cell transplantation) inactivating thymic T cellsby one or more of: irradiating the recipient mammal with, e.g., about700 rads of thymic irradiation, administering to the recipient one ormore doses of an anti T cell antibody, e.g., an anti-CD4 and/or ananti-CD8 monoclonal antibody, or administering to the recipient a shortcourse of an immunosuppressant, as is described in U.S. Ser. No.08/220,371 and further described below.

Other preferred embodiments include depleting or otherwise inactivatingnatural antibodies, e.g., by one or more of the administration of a drugwhich depletes or inactivates natural antibodies, e.g., deoxyspergualin;the administration of an anti-IgM antibody; or the adsorption of naturalantibodies from the recipient's blood, e.g., by contacting the hostsblood with donor antigen, e.g., by hemoperfusion of a donor organ, e.g.,a kidney or a liver, from the donor species.

In preferred embodiments the host or recipient is a post-natalindividual, e.g., an adult, or a child.

In preferred embodiments the method further includes the step ofidentifying a host or recipient which is at risk for an acquired immunedisorder, (e.g., a human at risk for AIDS), and which is thymus-functiondeficient and thus unable to produce a normal number of maturefunctional T cells or a sufficient number of mature functional T cellsfor a normal immune response.

Other methods can be combined with the methods disclosed herein topromote the acceptance of donor tissue by the recipient. For example,tolerance to donor tissue can be induced by inserting a nucleic acidwhich expresses a donor antigen, e.g., a donor MHC gene, into a cell ofthe recipient, e.g., a hematopoietic stem cell, and introducing thegenetically engineered cell into the recipient. For example, humanrecipient stem cells can be engineered to express a swine MHC gene, e.g.a swine class I or class II MHC gene, or both a class I and a class IIgene, and the cells implanted in a human recipient who will receiveswine thymic tissue. When inserted into a recipient primate, e.g., ahuman, expression of the donor MHC gene results in tolerance tosubsequent exposure to donor antigen, and can thus induce tolerance totissue from the donor. These methods and other methods which can becombined with the methods disclosed herein are discussed in Sachs, U.S.Ser. No. 08/126, 122, filed Sep. 23, 1993, and in Sachs, U.S. Ser. No.08/220,371, filed Mar. 29, 1994.

Methods of inducing tolerance, e.g., by the implantation ofhematopoietic stem cells, disclosed in Sachs, Cosimi, and Sykes, U.S.Ser. No. 07/838,595, filed Feb. 19, 1992, can also be combined with themethods disclosed herein.

Other methods of inducing tolerance can be combined with the methodsdisclosed herein to promote acceptance of donor tissue. For example,suppression of T cell help, which can be induced, e.g., by theadministration of a short course of high dose immunosuppressant, e.g.,cyclosporine, has been found to induce tolerance. In these methods, Tcell help is suppressed for a comparatively short period just subsequentto implantation of a graft, and does not require or include chronicimmunosuppression. These methods, as well as other methods which can becombined with the methods disclosed herein, are described in Sachs, U.S.Ser. No. 08/220,371, filed Mar. 29, 1994.

Other methods of promoting tolerance or promoting the acceptance ofdonor tissue, e.g., by altering levels of cytokine activity, orinhibiting Graft-versus-recipient-disease, are disclosed in Sachs,LeGuern, Sykes, and Blancho, U.S. Ser. No. 08/114,072, filed Aug. 30,1993. It has also been discovered that hematopoietic cells can be use toinduce tolerance to a graft.

Accordingly, in another aspect, the invention features, a method ofinducing immunological tolerance in a recipient mammal, e.g., a primate,e.g., a human, of a first species to a graft obtained from a donormammal of a second species, e.g., a discordant species e.g., adiscordant primate species. The method includes: prior to orsimultaneous with transplantation of the graft, introducing into therecipient mammal hematopoietic stem cells, e.g., bone marrow cells, orfetal liver or spleen cells, of the second species; (preferably, thehematopoietic stem cells home to a site in the recipient mammal);optionally, (preferably prior to introducing the hematopoietic stemcells into the recipient mammal), depleting, inactivating or inhibitingrecipient NK cells, e.g., by introducing into the recipient mammal anantibody capable of binding to natural killer (NK) cells of therecipient mammal, to prevent NK mediated rejection of the hematopoieticcells; and (optionally) implanting the graft in the recipient. As willbe explained in more detail below, the hematopoietic cells prepare therecipient for the graft that follows, by inducing tolerance at both theB-cell and T-cell levels. Preferably, hematopoietic cells are fetalliver or spleen, or bone marrow cells, including immature cells (i.e.,undifferentiated hematopoietic stem cells; these desired cells can beseparated out of the bone marrow prior to administration), or a complexbone marrow sample including such cells can be used.

One source of anti-NK antibody is anti-human thymocyte polyclonalanti-serum. A second, anti-mature T cell antibody can be administered aswell, which lyses T cells as well as NK cells. Lysing T cells isadvantageous for both bone marrow and xenograft survival. Anti-T cellantibodies are present, along with anti-NK antibodies, in anti-thymocyteanti-serum. Repeated doses of anti-NK or anti-T cell antibody may bepreferable. Monoclonal preparations can be used in the methods of theinvention.

Preferred embodiments include: the step of introducing into therecipient mammal, donor species-specific stromal tissue, preferablyhematopoietic stromal tissue, e.g., fetal liver or thymus; and the stepof prior to hematopoietic stem cell transplantation, introducing intothe recipient mammal an antibody capable of binding to mature T cells ofthe recipient mammal.

Preferred embodiments include those in which: the stromal tissue isintroduced simultaneously with, or prior to, the hematopoietic stemcells; the hematopoietic stem cells are introduced simultaneously with,or prior to, the antibody; the stromal tissue is introducedsimultaneously with, or prior to, the hematopoietic stem cells, and thehematopoietic stem cells are introduced simultaneously with, or priorto, the antibody.

Preferred embodiments include those in which: the same mammal of thesecond species is the donor of both the graft and the hematopoieticcells; the donor mammal is a swine, e.g., a miniature swine; theintroduction is by intravenous injection; and an anti-human thymocytepolyclonal anti-serum; obtained, e.g. from a horse or pig isadministered.

Other preferred embodiments include the step of (preferably prior tothymic tissue or hematopoietic stem cell transplantation) creatinghematopoietic space, e.g., by one or more of: irradiating the recipientmammal with low dose, e.g., between about 100 and 400 rads, whole bodyirradiation, the administration of a myelosuppressive drug, theadministration of a hematopoietic stem cell inactivating or depletingantibody, to deplete or partially deplete the bone marrow of therecipient.

Other preferred embodiments include (preferably prior to thymic tissueor hematopoietic stem cell transplantation) inactivating thymic T cellsby one or more of: irradiating the recipient mammal with, e.g., about700 rads of thymic irradiation, administering to the recipient one ormore doses of an anti T cell antibody, e.g., an anti-CD4 and/or ananti-CD8 monoclonal antibody, or administering to the recipient a shortcourse of an immunosuppressant, as is described in U.S. Ser. No.08/220,371 and further described below.

Other preferred embodiments include depleting or otherwise inactivatingnatural antibodies, e.g., by one or more of: the administration of adrug which depletes or inactivates natural antibodies, e.g.,deoxyspergualin; the administration of an anti-IgM antibody; or theadsorption of natural antibodies from the recipient's blood, e.g., bycontacting the hosts blood with donor antigen, e.g., by hemoperfusion ofa donor organ, e.g., a kidney or a liver, from the donor species.

Preferred embodiments include: (preferably prior to hematopoietic stemcell transplantation) depleting, inactivating, or inhibiting recipient Tcells, e.g., by introducing into the recipient an antibody capable ofbinding to mature T cells of the recipient.

Preferably the graft is obtained from a different organ than thehematopoietic stem cells.

Preferred embodiments include those in which: the primate is acynomolgus monkey; the primate is a human; the stromal tissue is fetalor neonatal liver; the stromal tissue is fetal or neonatal thymus; themammal is a swine; e.g., a miniature swine; the graft is a liver; thegraft is a kidney.

Other methods can be combined with the methods disclosed herein topromote the acceptance of the graft by the recipient. For example,tolerance to the xenogeneic thymic tissue can also be induced byinserting a nucleic acid which expresses a donor antigen, e.g., a donorMHC gene, into a cell of the recipient, e.g., a hematopoietic stem cell,and introducing the genetically engineered cell into the recipient. Forexample, human recipient stem cells can be engineered to express a swineclass I or class II MHC gene, or both a class I and II gene, and thecells implanted in a human recipient who will receive swine thymictissue. When inserted into a recipient primate, e.g., a human,expression of the donor MHC gene results in tolerance to subsequentexposure to donor antigen, and can thus induce tolerance to thymictissue from the donor. These methods, and other methods which can becombined with the methods disclosed herein, are discussed in Sachs, U.S.Ser. No. 08/126, 122, filed Sep. 23, 1993, and in Sachs, U.S. Ser. No.08/220,371, filed Mar. 29, 1994.

Methods of inducing tolerance, e.g., by the implantation ofhematopoietic stem cells, disclosed in Sachs, Cosimi, and Sykes, U.S.Ser. No. 07/838,595, filed Feb. 19, 1992, can also be combined with themethods disclosed herein.

Other methods of inducing tolerance may also be used to promoteacceptance of the xenogeneic thymic tissue. For example, suppression ofT cell help, which can be induced, e.g., by the administration of ashort course of high dose immunosuppressant, e.g., cyclosporine, hasbeen found to induce tolerance. In these methods, T cell help issuppressed for a comparatively short period just subsequent toimplantation of a graft, and does not require or include chronicimmunosuppression. These methods, as well as other methods which can becombined with the methods disclosed herein, are described in Sachs, U.S.Ser. No. 08/220,371, filed Mar. 29, 1994, hereby. incorporated byreference.

Other methods of promoting tolerance or promoting the acceptance ofgrafts, e.g., by altering levels of cytokine activity, are disclosed inSachs, LeGuern, Sykes, and Blancho, U.S. Ser. No. 08/114,072, filed Aug.30, 1993.

In another aspect, the invention features a method of inducingimmunological tolerance in a recipient mammal, e.g., a primate, e.g., ahuman to a graft obtained from a donor mammal of the same species. Themethod includes the following: (preferably prior to or simultaneous withtransplantation of the graft) introducing into the recipient mammalhematopoietic stem cells, e.g., bone marrow cells or fetal liver orspleen cells, obtained from a mammal (preferably, the hematopoietic stemcells home to a site in the recipient mammal); and, preferably,introducing the graft into the recipient.

Preferred embodiments include: the step of introducing into therecipient mammal, donor species-specific stromal tissue, preferablyhematopoietic stromal tissue, e.g., fetal liver or thymus; and prior tohematopoietic stem cell transplantation, depleting, inactivating orinhibiting recipient T cells, e.g., by introducing into the recipientmammal an antibody capable of binding to mature T cells of the recipientmammal.

Other preferred embodiments include the step of (preferably prior tothymic tissue or hematopoietic stem cell transplantation) creatinghematopoietic space, e.g., by one or more of: irradiating the recipientmammal with low dose, e.g., between about 100 and 400 rads, whole bodyirradiation, the administration of a myelosuppressive drug, theadministration of a hematopoietic stem cell inactivating or depletingantibody, to deplete or partially deplete the bone marrow of therecipient.

Other preferred embodiments include (preferably prior to thymic tissueor hematopoietic stem cell transplantation) inactivating thymic T cellsby one or more of: irradiating the recipient mammal with, e.g., about700 rads of thymic irradiation, administering to the recipient one ormore doses of an anti T cell antibody, e.g., an anti-CD4 and/or ananti-CD8 monoclonal antibody, or administering to the recipient a shortcourse of an immunosuppressant, as is described in U.S. Ser. No.08/220,371 and further described below.

Other preferred embodiments include depleting or otherwise inactivatingnatural antibodies, e.g., by one or more of: the administration of adrug which depletes or inactivates natural antibodies, e.g.,deoxyspergualin; the administration of an anti-IgM antibody; or theadsorption of natural antibodies from the recipient's blood, e.g., bycontacting the hosts blood with donor antigen, e.g., by hemoperfusion ofa donor organ, e.g., a kidney or a liver, from the donor species.

In other preferred embodiments; the method includes: (preferably priorto or at the time of introducing the thymic tissue into the recipient)depleting, inactivating or inhibiting recipient natural killer (NK)cells, e.g., by introducing into the recipient an antibody capable ofbinding to NK cells of the recipient, to prevent NK mediated rejectionof the thymic tissue; (preferably prior to or at the time of introducingthe thymic tissue into the recipient) depleting, inactivating orinhibiting host T cell function, e.g., by introducing into the recipientan antibody capable of binding to T cells of the recipient.

Other methods can be combined with-the methods disclosed herein topromote tolerance to a graft. Methods of inducing tolerance, e.g., bythe implantation of hematopoietic stem cells, disclosed in Sachs,Cosimi, and Sykes, U.S. Ser. No. 07/838,595, filed Feb. 19, 1992, canalso be combined with the methods disclosed herein.

Other methods of inducing tolerance may also be used to promoteacceptance of donor tissue. For example, suppression of T cell help,which can be induced, e.g., by the administration of a short course ofhigh dose immunosuppressant, e.g., cyclosporine, has been found toinduce tolerance. In these methods, T cell help is suppressed for acomparatively short period just subsequent to implantation of a graft,and does not require or include chronic immunosuppression. Thesemethods, as well as other methods which can be combined with the methodsdisclosed herein, are described in Sachs, U.S. Ser. No. 08/220,371,filed Mar. 29, 1994.

Other methods of promoting tolerance or promoting the acceptance ofgrafts, e.g., by altering levels of cytokine activity, are disclosed inSachs, LeGuern, Sykes, and Blancho, U.S. Ser. No. 08/114,072, filed Aug.30, 1993.

The invention further provides several methods of inducing tolerance toforeign antigens, e.g., to antigens on allogeneic or xenogeneic tissueor organ grafts. These methods can be used individually or incombination with one another. For example, it has been found that theshort-term administration of a help reducing agent, e.g., a short highdose course of cyclosporine A (CsA), can significantly prolong graftacceptance. The short term help reduction-methods of the invention canbe combined with one or more other methods for prolonging graftacceptance. For example, a short course of high dose cyclosporinetreatment to induce tolerance to unmatched donor class I and other minorunmatched donor antigens can be combined with implantation ofretrovirally transformed bone marrow cells to induce tolerance tounmatched donor class II. A short course of high dose cyclosporineadministered to induce tolerance to unmatched donor class I and otherminor antigens can also be combined with implantation of donor bonemarrow cells to induce tolerance to unmatched donor class II.

Accordingly, the invention features, in one aspect, a method of inducingtolerance in a recipient mammal, e.g., a primate, e.g., a human, to anallograft from a donor primate including: implanting the graft in therecipient; and administering to the recipient a short course of helpreducing treatment, e.g., a short course of high dose cyclosporine. Theshort course of help reducing treatment is generally administered atabout the time the graft is introduced into the recipient.

Preferably, the recipient is mismatched at a first locus which affectsgraft rejection, e.g., an MHC class I or II locus, or a minor antigenlocus, and matched, or tolerant of a mismatch, at a second locus whichaffects graft rejection, e.g., an MHC class I or II locus, or a minorantigen locus. Matching at the second locus can be achieved by selectionof a recipient or donor of the appropriate genotype. The recipient canbe rendered tolerant of a mismatch at the second locus by any method oftolerance induction, e.g., by administering donor bone marrow tissue tothe recipient to induce tolerance to donor antigens expressed on thedonor bone marrow, by expressing an MHC antigen of the donor from a stemcell of the recipient to induce tolerance to the donor antigen, or byaltering the immunological properties of the graft, e.g., by masking,cleaving, or otherwise modifying cell surface molecules on the graft. Inpreferred embodiments, any of the methods which can be used to match orinduce tolerance to the second locus can be used to match or inducetolerance to a third locus which affects graft rejection, e.g., an MHCclass I or II locus, or a minor antigen locus.

In preferred embodiments, the recipient and donor are matched at a classII locus and the short course of help reducing treatment inducestolerance to unmatched class I and/or minor antigens on the graft. Inpreferred embodiments, tolerance to a class II antigen is induced by amethod other than a short course of a help reducing treatment, and theshort course of help reducing treatment induces tolerance to unmatchedclass I and minor antigens on the graft.

In preferred embodiments, the duration of the short course of helpreducing treatment is approximately equal to or is less than the periodrequired for mature T cells of the recipient species to initiaterejection of an antigen after first being stimulated by the antigen (inhumans this is usually 8-12 days, preferably about 10 days); in morepreferred embodiments, the duration is approximately equal to or is lessthan two, three, four, five, or ten times the period required for matureT cells of the recipient to initiate rejection of an antigen after firstbeing stimulated by the antigen.

In other preferred embodiments, the short course of help reducingtreatment is administered in the absence of a treatment which stimulatesthe release of a cytokine by mature T cells in the recipient, e.g. inthe absence of a steroid drug in a sufficient concentration tocounteract the desired effect of the help reducing treatment, e.g., inthe absence of Prednisone(17,21-dihydroxypregna-1,4-diene-3,11,20-trione) at a concentrationwhich stimulates the release of a cytokine by mature T cells in therecipient. In preferred embodiments, the short course of help reducingtreatment is administered in the absence of a steroid drug, e.g., in theabsence of Prednisone.

In preferred embodiments: the help reducing treatment is begun before orat about the time the graft is introduced; the short course isperioperative, or the short course is postoperative; or the donor andrecipient are class I matched.

Methods of inducing tolerance by a short-term administration of a helpreducing agent, e.g., a short high dose course of cyclosporine A (CsA),can be combined with other methods for inducing tolerance, e.g., methodsfor the implantation of transduced bone marrow cells to induce toleranceto an antigen, e.g., the methods described in U.S. Ser. No. 008/126,122,filed on Sep. 23, 1993.

Accordingly, in another aspect, the invention features a method ofinducing tolerance in a recipient mammal, e.g., a primate, e.g., ahuman, of a first species to a graft from a mammal, e.g., a swine, e.g.,a miniature swine, of a second species, which graft preferably expressesa major histocompatibility complex (MHC) antigen. The method includesinserting DNA encoding an MHC antigen of the second species into ahematopoietic stem cell, e.g., a bone marrow hematopoietic stem cell, ofthe recipient mammal; allowing the MHC antigen encoding DNA to beexpressed in the recipient; preferably, implanting the graft in therecipient; and, preferably, administering to the recipient a shortcourse of help reducing treatment, e.g., a short course of high dosecyclosporine treatment. The short course of help reducing treatment isgenerally administered at about the time the graft is introduced intothe recipient.

In preferred embodiments, the short course of help reducing treatmentinduces tolerance to unmatched class I and/or minor antigens on a graftwhich is introduced into the recipient subsequent to expression of theMHC antigen.

In preferred embodiments, the duration of the short course of helpreducing treatment is approximately equal to or is less than the periodrequired for mature T cells of the recipient species to initiaterejection of an antigen after first being stimulated by the antigen; inmore preferred embodiments, the duration is approximately equal to or isless than two, three, four, five, or ten times the period required for amature T cell of the recipient species to initiate rejection of anantigen after first being stimulated by the antigen.

In other preferred embodiments, the short course of help reducingtreatment is administered in the absence of a treatment which stimulatesthe release of a cytokine by mature T cells in the recipient, e.g., inthe absence of a steroid drug in a sufficient concentration tocounteract the desired effect of the help reducing treatment, e.g., inthe absence of Prednisone(17,21-dihydroxypregna-1,4-diene-3,11,20-trione) at a concentrationwhich stimulates the release of a cytokine by mature T cells in therecipient. In preferred embodiments, the short course of help reducingtreatment is administered in the absence of a steroid drug, e.g., in theabsence of Prednisone.

In preferred embodiments: the help reducing treatment is begun before orat about the time the graft is introduced; the short course isperioperative; or the short course is postoperative.

Preferred embodiments include those in which: the cell is removed fromthe recipient mammal prior to the DNA insertion and returned to therecipient mammal after the DNA insertion; the DNA is obtained from theindividual mammal from which the graft is obtained; the DNA is obtainedfrom an individual mammal which is syngeneic with the individual mammalfrom which the graft is obtained; the DNA is obtained from an individualmammal which is MHC matched, and preferably identical, with theindividual mammal from which the graft is obtained; the DNA includes anMHC class I gene; the DNA includes an MHC class II gene; the DNA isinserted into the cell by transduction, e.g., by a retrovirus, e.g., bya Moloney-based retrovirus; and the DNA is expressed in bone marrowcells and/or peripheral blood cells of the recipient for at least 14,preferably 30, more preferably 60, and most preferably 120 days, afterthe DNA is introduced into the recipient.

Other preferred embodiments include: the step of, prior to hematopoieticstem cell transplantation, creating hematopoietic space, e.g., byirradiating the recipient mammal with low dose, e.g., between about 100and 400 rads, whole body irradiation to deplete or partially deplete thebone marrow of the recipient; inactivating thymic T cells by one or moreof: prior to hematopoietic stem cell transplantation, irradiating therecipient mammal with, e.g., about 700 rads of thymic irradiation, oradministering to the recipient a short course of an immunosuppressant,as is described herein.

Other preferred embodiments include: the step of, prior to implantationof a graft, depleting natural antibodies from the blood of the recipientmammal, e.g., by hemoperfusing an organ, e.g., a liver or a kidney,obtained from a mammal of the second species. (In organ hemoperfusionantibodies in the blood bind to antigens on the cell surfaces of theorgan and are thus removed from the blood.)

In other preferred embodiments: the method further includes, prior tohematopoietic stem cell transplantation, introducing into the recipientan antibody capable of binding to mature T cells of said recipientmammal.

Other preferred embodiments further include the step of introducing intothe recipient a graft obtained from the donor, e.g., a liver or akidney.

In another aspect, the invention features a method of inducing tolerancein a recipient mammal, preferably a primate, e.g., a human, to a graftobtained from a donor of the same species, which graft preferablyexpresses an MHC antigen. The method includes: inserting DNA encoding anMHC antigen of the donor into a hematopoietic stem cell, e.g., bonemarrow hematopoietic stem cell, of the recipient; allowing the MHCantigen encoding DNA to be expressed in the recipient; preferably,implanting the graft in the recipient; and, preferably, administering tothe recipient a short course of help reducing treatment, e.g., a shortcourse of high dose cyclosporine. The short course of help reducingtreatment is generally administered at about the time the graft isintroduced into the recipient.

In preferred embodiments, the short course of help reducing treatmentinduces tolerance to unmatched class I and/or minor antigens on a graftwhich is introduced into the recipient subsequent to expression of theMHC antigen.

In preferred embodiments, the duration of the short course of helpreducing treatment is approximately equal to or is less than the periodrequired for mature T cells of the recipient species to initiaterejection of an antigen after first being stimulated by the antigen; inmore preferred embodiments, the duration is approximately equal to or isless than two, three, four, five, or ten times the period required for amature T cell of the recipient species to initiate rejection of anantigen after first being stimulated by the antigen.

In other preferred embodiments, the short course of help reducingtreatment is administered in the absence of a treatment which stimulatesthe release of a cytokine by mature T cells in the recipient, e.g., inthe absence of a steroid drug in a sufficient concentration tocounteract the desired effect of the help reducing treatment, e.g., inthe absence of Prednisone(17,21-dihydroxypregna-1,4-diene-3,11,20-trione) at a concentrationwhich stimulates the release of a cytokine by mature T cells in therecipient. In preferred embodiments, the short course of help reducingtreatment is administered in the absence of a steroid drug, e.g., in theabsence of Prednisone

In preferred embodiments: the help reducing treatment is begun before orat about the time the graft is introduced; the short course isperioperative, or the short course is postoperative; or the donor andrecipient are class I matched.

Preferred embodiments include those in which: the cell is removed fromthe recipient prior to the DNA insertion and returned to the recipientafter the DNA insertion; the DNA includes a MHC class I gene; the DNAincludes a MHC class II gene; the DNA is inserted into the cell bytransduction, e.g. by a retrovirus, e.g., by a Moloney-based retrovirus;and the DNA is expressed in bone marrow cells and/or peripheral bloodcells of the recipient at least 14, preferably 30, more preferably 60,and most preferably 120 days, after the DNA is introduced into therecipient.

In other preferred embodiments: the method further includes, prior tohematopoietic stem cell transplantation, introducing into the recipientan antibody capable of binding to mature T cells of said recipientmammal.

In preferred embodiments the graft is a liver or a kidney.

Other preferred embodiments include: the step of, prior to hematopoieticstem cell transplantation, creating hematopoietic space, e.g., byirradiating the recipient mammal with low dose, e.g., between about 100and 400 rads, whole body irradiation to deplete or partially deplete thebone marrow of the recipient; inactivating thymic T cells by one or moreof: prior to hematopoietic stem cell transplantation, irradiating therecipient mammal with, e.g., about 700 rads of thymic irradiation, oradministering to the recipient a short course of an immunosuppressant,as is described herein.

Other preferred embodiments include: the step of, prior to implantationof a graft, depleting natural antibodies from the blood of the recipientmammal, e.g., by hemoperfusing an organ, e.g., a liver or a kidney,obtained from a mammal of the second species. (In organ hemoperfusionantibodies in the blood bind to antigens on the cell surfaces of theorgan and are thus removed from the blood.)

Methods of inducing tolerance with a short-term administration of a helpreducing agent, e.g., a short high dose course of cyclosporine A (CsA),can be combined with other methods for inducing tolerance, e.g., methodsof inducing tolerance which use the implantation of donor stem cells toinduce tolerance to an antigen, e.g., the methods described in U.S. Ser.No. 07/838,595, filed Feb. 19, 1992.

Accordingly, in another aspect, the invention features a method ofinducing tolerance in a recipient mammal of a first species, e.g., aprimate, e.g., a human, to a graft obtained from a mammal of a second,preferably discordant species, e.g., a swine, e.g., a miniature swine,or a discordant primate species. The method includes: preferably priorto or simultaneous with transplantation of the graft, introducing, e.g.,by intravenous injection, into the recipient mammal, hematopoietic stemcells, e.g., bone marrow cells or fetal liver or spleen cells, of thesecond species (preferably the hematopoietic stem cells home to a sitein the recipient mammal); (optionally) inactivating the natural killercells of the recipient mammal, e.g., by prior to introducing thehematopoietic stem cells into the recipient mammal, introducing into therecipient mammal an antibody capable of binding to natural killer cellsof said recipient mammal; preferably, implanting the graft in therecipient; and, preferably, administering to the recipient a shortcourse of help reducing treatment, e.g., a short course of high dosecyclosporine. The short course of help reducing treatment is generallyadministered at the time at the graft is introduced into the recipient.

In preferred embodiments, the short course of help reducing treatmentinduces tolerance to unmatched class I and/or minor antigens on thegraft which is introduced into the recipient.

In preferred embodiments, the duration of the short course of helpreducing treatment is approximately equal to or is less than the periodrequired for mature T cells of the recipient species to initiaterejection of an antigen after first being stimulated by the antigen; inmore preferred embodiments, the duration is approximately equal to or isless than two, three, four, five, or ten times, the period required fora mature T cell of the recipient species to initiate rejection of anantigen after first being stimulated by the antigen.

In other preferred embodiments, the short course of help reducingtreatment is administered in the absence of a treatment which stimulatesthe release of a cytokine by mature T cells in the recipient, e.g., inthe absence of a steroid drug in a sufficient concentration tocounteract the desired effect of the help reducing treatment, e.g., inthe absence of Prednisone(17,21-dihydroxypregna-1,4-diene-3,11,20-trione) at a concentrationwhich stimulates the release of a cytokine by mature T cells in therecipient. In preferred embodiments, the short course of help reducingtreatment is administered in the absence of a steroid drug, e.g., in theabsence of Prednisone.

In preferred embodiments: the help reducing treatment is begun before orat about the time the graft is introduced; or the short course isperioperative, the short course is postoperative.

As will be explained in more detail below, the hematopoietic cellsprepare the recipient for the graft that follows, by inducing toleranceat both the B-cell and T-cell levels. Preferably, hematopoietic cellsare fetal liver or spleen, or bone marrow cells, including immaturecells (i.e., undifferentiated hematopoietic stem cells; these desiredcells can be separated out of the bone marrow prior to administration),or a complex bone marrow sample including such cells can be used.

One source of anti-NK antibody is anti-human thymocyte polyclonalanti-serum. As is discussed below, preferably, a second anti-mature Tcell antibody can be administered as well, which lyses T cells as wellas NK cells. Lysing T cells is advantageous for both bone marrow andxenograft survival. Anti-T cell antibodies are present, along withanti-NK antibodies, in anti-thymocyte anti-serum. Repeated doses ofanti-NK or anti-T cell antibody may be preferable. Monoclonalpreparations can be used in the methods of the invention.

Other preferred embodiments include: the step of introducing into therecipient mammal, donor species-specific stromal tissue, preferablyhematopoietic stromal tissue, e.g., fetal liver or thymus. In preferredembodiments: the stromal tissue is introduced simultaneously with, orprior to, the hematopoietic stem cells; the hematopoietic stem cells areintroduced simultaneously with, or prior to, the antibody.

Other preferred embodiments include those in which: the same mammal ofthe second species is the donor of one or both the graft and thehematopoietic cells; and the antibody is an anti-human thymocytepolyclonal anti-serum, obtained, e.g., from a horse or pig.

Other preferred embodiments include: the step of, prior to hematopoieticstem cell transplantation, creating hematopoietic space, e.g., byirradiating the recipient mammal with low dose, e.g., between about 100and 400 rads, whole body irradiation to deplete or partially deplete thebone marrow of the recipient; inactivating thymic T cells by one or moreof: prior to hematopoietic stem cell transplantation, irradiating therecipient mammal with, e.g., about 700 rads of thymic irradiation, oradministering to the recipient a short course of an immunosuppressant,as is described herein.

Other preferred embodiments include: the step of, prior to hematopoieticstem cell transplantation, depleting natural antibodies from the bloodof the recipient mammal, e.g., by hemoperfusing an organ, e.g., a liveror a kidney, obtained from a mammal of the second species. (In organhemoperfusion antibodies in the blood bind to antigens on the cellsurfaces of the organ and are thus removed from the blood.)

In other preferred embodiments: the method further includes, prior tohematopoietic stem cell transplantation, introducing into the recipientan antibody capable of binding to mature T cells of said recipientmammal.

In other preferred embodiments: the method further includes inactivatingT cells of the recipient, e.g., by, prior to introducing thehematopoietic stem cells into the recipient, introducing into therecipient an antibody capable of binding to T cells of the recipient.

In preferred embodiments, the method includes the step of introducinginto the recipient a graft obtained from the donor which is obtainedfrom a different organ than the hematopoietic stem cells, e.g., a liveror a kidney.

In another aspect, the invention features a method of inducing tolerancein a recipient mammal, preferably a primate, e.g., a human, to a graftobtained from a donor, e.g., of the same species. The method includes:preferably prior to or simultaneous with transplantation of the graft,introducing, e.g., by intravenous injection, into the recipient,hematopoietic stem cells, e.g., bone marrow cells or fetal liver orspleen cells, of a mammal, preferably the donor (preferably thehematopoietic stem cells home to a site in the recipient); (optionally),inactivating T cells of the recipient, e.g., by, prior to introducingthe hematopoietic stem cells into the recipient, introducing into therecipient an antibody capable of binding to T cells of the recipient;preferably, implanting the graft in the recipient; and, preferably,administering to the recipient a short course of help reducingtreatment, e.g., a short course of high dose cyclosporine. The shortcourse of help reducing treatment is generally administered at the timethe graft is introduced into the recipient.

In preferred embodiments, the short course of help reducing treatmentinduces tolerance to unmatched class I and minor antigens on the graftwhich is introduced into the recipient.

In preferred embodiments, the duration of the short course of helpreducing treatment is approximately equal to or is less than the periodrequired for mature T cells of the recipient species to initiaterejection of an antigen after first being stimulated by the antigen; inmore preferred embodiments, the duration is approximately equal to or isless than two, three, four, five, or ten times the period required for amature T cell of the recipient species to initiate rejection of anantigen after first being stimulated by the antigen.

In other preferred embodiments, the short course of help reducingtreatment is administered in the absence of a treatment which stimulatesthe release of a cytokine by mature T cells in the recipient, e.g., inthe absence of a steroid drug in a sufficient concentration tocounteract the desired effect of the help reducing treatment, e.g., inthe absence of Prednisone(17,21-dihydroxypregna-1,4-diene-3,11,20-trione) at a concentrationwhich stimulates the release of a cytokine by mature T cells in therecipient. In preferred embodiments, the short course of help reducingtreatment is administered in the absence of a steroid drug, e.g., in theabsence of Prednisone

In preferred embodiments: the help reducing treatment is begun before orat about the time the graft is introduced; the short course isperioperative, the short course is postoperative; the donor andrecipient are class I matched.

In preferred embodiments, the hematopoietic stem cells are introducedsimultaneously with, or prior to administration of the antibody; theantibody is an antihuman thymocyte polyclonal anti-serum; and theanti-serum is obtained from a horse or pig.

Other preferred embodiments include: the further step of, prior tohematopoietic stem cell transplantation, inactivating or depleting NKcells of the recipient, e.g., by introducing into the recipient mammalan antibody capable of binding to NK cells of the recipient mammal; andthose in which the same individual is the donor of both the graft andthe bone marrow.

Other preferred embodiments include: the step of, prior to hematopoieticstem cell transplantation, creating hematopoietic space, e.g., byirradiating the recipient mammal with low dose, e.g., between about 100and 400 rads, whole body irradiation to deplete or partially deplete thebone marrow of the recipient; inactivating thymic T cells by one or moreof, prior to hematopoietic stem cell transplantation, irradiating therecipient mammal with, e.g., about 700 rads of thymic irradiation, oradministering to the recipient a short course of an immunosuppressant,as is described herein.

Other preferred embodiments include: the further step of, prior to bonemarrow transplantation, adsorbing natural antibodies from the blood ofthe recipient by hemoperfusing an organ, e.g., the liver, or a kidney,obtained from the donor.

Preferred embodiments include: the step of introducing into therecipient mammal, donor species specific stromal tissue, preferablyhematopoietic stromal tissue, e.g., fetal liver or thymus.

In preferred embodiments, the method includes the step of introducinginto the recipient, a graft which is obtained from a different organthan the hematopoietic stem cells, e.g., a liver or a kidney.

Methods of inducing tolerance with short-term administration of a helpreducing agent, e.g., a short high dose course of cyclosporine A (CsA),can be combined with yet other methods for inducing tolerance, e.g.,with: methods which use the implantation of a xenogeneic thymic graft toinduce tolerance, e.g., the methods described in U.S. Ser. No. 08/163,912 filed on Dec. 7, 1993; methods of increasing the level of theactivity of a tolerance promoting or GVHD inhibiting cytokine ordecreasing the level of activity of a tolerance inhibiting or GVHDpromoting cytokine, e.g., the methods described in U.S. Ser. No.08/114,072, filed Aug. 30, 1993; methods of using cord blood cells toinduce tolerance, e.g., the methods described in U.S. Ser. No.08/150,739 filed Nov. 10, 1993; and the methods for inducing tolerancedisclosed in Sykes and Sachs, PCT/US94/01616, filed Feb. 14, 1994.

It has also been discovered that a short course of an immunosuppressant,e.g., cyclosporine, can be used to diminish or inhibit T cell activitywhich would otherwise promote the rejection of an allograft orxenograft.

Accordingly, in another aspect, the invention features a method ofdiminishing or inhibiting T cell activity, preferably the activity ofthymic or lymph node T cells, in a recipient mammal, e.g., a primate,e.g., a human, which receives a graft from a donor mammal. The methodincludes, inducing tolerance to the graft; administering to therecipient a short course of an immunosuppressive agent, e.g.,cyclosporine, sufficient to inactivate T cells, preferably thymic orlymph node T cells; and preferably transplanting the graft into therecipient.

Tolerance to the graft can be induced by any method, e.g., by any of themethods discussed herein. For example, tolerance can be induced by theadministration of donor allogeneic or xenogeneic hematopoietic stemcells, the administration of genetically engineered autologous stemcells, by the administration of a short course of a help reducing agent,or by altering the immunological properties of the graft, e.g., bymasking, cleaving, or otherwise modifying cell surface molecules of thegraft.

In preferred embodiments the duration of the short course ofimmunosuppressive agent is: approximately equal to 30 days;approximately equal to or less than 8-12 days, preferably about 10 days;approximately equal to or less than two, three, four, five, or ten timesthe 8-12 or 10 day period.

In preferred embodiments: the short course is begun before or at aboutthe time the treatment to induce tolerance is begun, e.g., at about thetime, xenogeneic, allogeneic, genetically engineered syngeneic, orgenetically engineered autologous stem cells are introduced into therecipient; the short course begins on the day the treatment to inducetolerance is begun, e.g., on the day, xenogeneic, allogeneic,genetically engineered syngeneic, or genetically engineered autologousstem cells are introduced into the recipient; the short course beginswithin 1, 2, 4, 6, 8, or 10 days before or after the treatment to inducetolerance is begun, e.g., within 1, 2, 4, 6, 8, or 10 days before orafter xenogeneic, allogeneic, genetically engineered syngeneic, orgenetically engineered autologous stem cells are introduced into therecipient.

In other preferred embodiments: the short course of an immunosuppressiveis administered in conjunction with an anti-T cell antibody; the shortcourse of an immunosuppressive is sufficient to inactivate T cells,e.g., thymic or lymph node T cells, which would not be inactivated byantibody-based inactivation of T cells, e.g., inactivation byintravenous administrations of ATG antibody, or similar, preparations.

In preferred embodiments: the recipient mammal is other than a mouse orrat.

Methods of inactivating T cells, preferably thymic or lymph node Tcells, of the invention can be combined with methods of inducingtolerance in which the inactivation of T cells is desirable. The anti-Tcell methods of the invention can be used in place of, or in additionto, methods for the inactivation of T cells called for, or useful insuch methods of inducing tolerance. For example, anti-thymic or lymphnode T cell methods of the invention can be used with methods for theimplantation of transduced bone marrow cells to induce tolerance to anantigen, e.g., the methods described in U.S. Ser. No. 008/126,122, filedon Sep. 23, 1993.

Accordingly, in another aspect, the invention features a method ofpromoting, in a recipient mammal of a first species, the acceptance of agraft from a donor mammal of a second species, which graft, preferably,expresses a major histocompatibility complex (MHC) antigen. The methodincludes inserting DNA encoding an MHC antigen of the second speciesinto a hematopoietic stem cell, e.g., a bone marrow hematopoietic stemcell, of the recipient mammal; allowing the MHC antigen encoding DNA tobe expressed in the recipient; and, preferably, administering to therecipient a short course of an immunosuppressive agent, e.g., a shortcourse of cyclosporine treatment, sufficient to inactivate recipient Tcells, preferably thymic or lymph node T cells. (Thymic or lymph node Tcells might otherwise inhibit the survival of the graft or engineeredcells.)

In preferred embodiments, the duration of the short course ofimmunosuppressive agent is: approximately equal to 30 days;approximately equal to or less than 8-12 days, preferably about 10 days;approximately equal to or less than two, three, four, five, or ten timesthe 8-12 or 10 day period.

In preferred embodiments: the recipient mammal is a primate, e.g., ahuman, and the donor mammal is a swine, e.g., a miniature swine.

In preferred embodiments: the short course is begun before or at aboutthe time genetically engineered stem cells are introduced into therecipient; the short course begins on the day the genetically engineeredstem cells are introduced into the recipient; the short course beginswithin 1, 2, 4, 6, 8, or 10 days before or after the geneticallyengineered stem cells are introduced into the recipient.

In other preferred embodiments: the short course of an immunosuppressiveagent is administered in conjunction with an anti-T cell antibody; theshort course of immunosuppressive is sufficient to inactivate T cells,e.g., thymic or lymph node T cells, which would not be inactivated byantibody-based inactivation of T cells, e.g., inactivation byintravenous administrations of ATG, or similar, antibody preparations.

Preferred embodiments include those in which: the cell is removed fromthe recipient mammal prior to the DNA insertion and returned to therecipient mammal after the DNA insertion; the DNA is obtained from theindividual mammal from which the graft is obtained; the DNA is obtainedfrom an individual mammal which is syngeneic with the individual mammalfrom which the graft is obtained; the DNA is obtained from an individualmammal which is MHC matched, and preferably identical, with theindividual mammal from which the graft is obtained; the DNA includes anMHC class I gene; the DNA includes an MHC class II gene; the DNA isinserted into the cell by transduction, e.g., by a retrovirus, e.g., bya Moloney-based retrovirus; and the DNA is expressed in bone marrowcells and/or peripheral blood cells of the recipient for at least 14,preferably 30, more preferably 60, and most preferably 120 days, afterthe DNA is introduced into the recipient.

Other preferred embodiments include: the step of, prior to hematopoieticstem cell implantation, creating hematopoietic space in the recipient soas to promote engraftment and survival of the implanted stem cells,e.g., by irradiating the recipient mammal with low dose, e.g., betweenabout 100 and 400 rads, whole body irradiation to deplete or partiallydeplete the bone marrow of the recipient.

In preferred embodiments, the method further includes the administrationof thymic irradiation to the recipient, e.g., 700 rads of thymicirradiation.

Other preferred embodiments include: the step of depleting naturalantibodies from the blood of the recipient mammal, e.g., byhemoperfusing an organ, e.g., a liver or a kidney, obtained from amammal of the second species. (In organ hemoperfusion antibodies in theblood bind to antigens on the cell surfaces of the organ and are thusremoved from the blood.)

Other preferred embodiments further include the step of introducing intothe recipient a graft obtained from the donor, e.g., a liver or akidney.

In another aspect, the invention features a method of promoting, in arecipient mammal, preferably a primate, e.g., a human, acceptance of agraft obtained from a donor of the same species, which graft expressesan MHC antigen. The method includes: inserting DNA encoding an MHCantigen of the donor into a hematopoietic stem cell, e.g., a bone marrowhematopoietic stem cell, of the recipient; allowing the MHC antigenencoding DNA to be expressed in the recipient; and, preferably,administering to the recipient a short course of an immunosuppressiveagent, e.g., a short course of cyclosporine treatment, sufficient toinactivate recipient T cells, preferably thymic or lymph node T cells.(Thymic or lymph node T cells might otherwise inhibit the survival ofthe graft or engineered cells.)

In preferred embodiments, the duration of the short course ofimmunosuppressive agent is: approximately equal to 30 days;approximately equal to or less than 8-12 days, preferably about 10 days;approximately equal to or less than two, three, four, five, or ten timesthe 8-12 or 10 day period.

In preferred embodiments: the short course is begun before or at aboutthe time genetically engineered stem cells are introduced into therecipient; the short course begins on the day the genetically engineeredstem cells are introduced into the recipient; the short course beginswithin 1, 2, 4, 6, 8, or 10 days before or after the geneticallyengineered stem cells are introduced into the recipient.

In other preferred embodiments: the short course of an immunosuppressiveagent is administered in conjunction with an anti-T cell antibody; theshort course of immunosuppressive is sufficient to inactivate T cells,e.g., thymic or lymph node T cells, which would not be inactivated byantibody-based inactivation of T cells, e.g., inactivation byintravenous administrations of ATG, or similar, antibody preparations.

Preferred embodiments include those in which: the cell is removed fromthe recipient prior to the DNA insertion and returned to the recipientafter the DNA insertion; the DNA includes a MHC class I gene; the DNAincludes a MHC class II gene; the DNA is inserted into the cell bytransduction, e.g. by a retrovirus, e.g., by a Moloney-based retrovirus;and the DNA is expressed in bone marrow cells and/or peripheral bloodcells of the recipient at least 14, preferably 30, more preferably 60,and most preferably 120 days, after the DNA is introduced into therecipient.

Other preferred embodiments include: the step of, prior to hematopoieticstem cell implantation, creating hematopoietic space in the recipient soas to promote engraftment and survival of the implanted stem cells,e.g., by irradiating the recipient mammal with low dose, e.g., betweenabout 100 and 400 rads, whole body irradiation to deplete or partiallydeplete the bone marrow of the recipient.

In preferred embodiments, the method further includes the administrationof thymic irradiation to the recipient, e.g., 700 rads of thymicirradiation.

Other preferred embodiments include: the step of depleting naturalantibodies from the blood of the recipient mammal, e.g., byhemoperfusing an organ, e.g., a liver or a kidney, obtained from amammal of the second species. (In organ hemoperfusion antibodies in theblood bind to antigens on the cell surfaces of the organ and are thusremoved from the blood.)

Other preferred embodiments further include the step of introducing intothe recipient a graft obtained from the donor, e.g., a liver or akidney.

Methods of inactivating T cells, preferably thymic or lymph node Tcells, of the invention can be combined with methods of inducingtolerance which use the implantation of donor stem cells to inducetolerance to an antigen, e.g., the methods described in U.S. Ser. No.07/838,595, filed Feb. 19, 1992, hereby incorporated by reference.

Accordingly, in another aspect, the invention features a method ofpromoting, in a recipient mammal of a first species, e.g., a primate,e.g., a human, acceptance of a graft obtained from a mammal of a second,preferably discordant species, e.g., a swine, e.g., a miniature swine,or a discordant primate species. The method includes: introducing, e.g.,by intravenous injection, into the recipient mammal, hematopoietic stemcells, e.g., bone marrow cells or fetal liver or spleen cells, of thesecond species (preferably the hematopoietic stem cells home to a sitein the recipient mammal); (optionally) inactivating natural killer cellsof the recipient mammal, e.g., by, prior to introducing thehematopoietic stem cells into the recipient mammal, introducing into therecipient mammal an antibody capable of binding to natural killer cellsof said recipient mammal; (optionally) inactivating T cells of therecipient mammal, e.g., by, prior to introducing the hematopoietic stemcells into the recipient mammal, introducing into the recipient mammalan antibody capable of binding to T cells of the recipient mammal; and,preferably, administering to the recipient a short course of animmunosuppressive agent, e.g., a short course of cyclosporine treatment,sufficient to inactivate recipient T cells, preferably thymic or lymphnode T cells. (Thymic or lymph node T cells might otherwise inhibit theengraftment or survival of the engineered cells.)

In preferred embodiments, the duration of the short course ofimmunosuppressive agent is: approximately equal to 30 days;approximately equal to or less than 8-12 days, preferably about 10 days;approximately equal to or less than two, three, four, five, or ten timesthe 8-12 or 10 day period mentioned above.

In preferred embodiments: the short course is begun before or at aboutthe time stem cells are introduced into the recipient; the short coursebegins on the day the stem cells are introduced into the recipient; theshort course begins within 1, 2, 4, 6, 8, or 10 days before or after thestem cells are introduced into the recipient.

In other preferred embodiments: the short course of an immunosuppressiveagent is administered in conjunction with one or both of an anti-T cellantibody, or thymic irradiation, e.g., 700 rads of thymic irradiation;the short course of immunosuppressive is sufficient to inactivate Tcells, e.g., thymic or lymph node T cells, which would not beinactivated by antibody-based inactivation of T cells, e.g.,inactivation by intravenous administrations of ATG antibodypreparations.

As will be explained in more detail below, the hematopoietic cellsprepare the recipient for the graft that follows, by inducing toleranceat both the B-cell and T-cell levels. Preferably, hematopoietic cellsare fetal liver or spleen, or bone marrow cells, including immaturecells (i.e., undifferentiated hematopoietic stem cells; these desiredcells can be separated out of the bone marrow prior to administration),or a complex bone marrow sample including such cells can be used.

One source of anti-NK antibody is anti-human thymocyte polyclonalanti-serum. As is discussed below, preferably, a second anti-mature Tcell antibody can be administered as well, which lyses T cells as wellas NK cells. Lysing T cells is advantageous for both bone marrow andxenograft survival. Anti-T cell antibodies are present, along withanti-NK antibodies, in anti-thymocyte anti-serum. Repeated doses ofanti-NK or anti-T cell antibody may be preferable. Monoclonalpreparations can be used in the methods of the invention.

Other preferred embodiments include: the step of introducing into therecipient mammal, donor species-specific stromal tissue, preferablyhematopoietic stromal tissue, e.g., fetal liver or thymus. In preferredembodiments: the stromal tissue is introduced simultaneously with, orprior to, the hematopoietic stem cells; the hematopoietic stem cells areintroduced simultaneously with, or prior to, an anti-NK or T cellantibody.

Other preferred embodiments include those in which: the same mammal ofthe second species is the donor of one or both the graft and thehematopoietic cells; and the anti-T or anti-NK cell antibody is ananti-human thymocyte polyclonal anti-serum, obtained, e.g., from a horseor pig.

Other preferred embodiments include: the step of, prior to hematopoieticstem cell transplantation, creating hematopoietic space in the recipientso as to promote engraftment and survival of the implanted stem cells,e.g., by irradiating the recipient mammal with low dose, e.g., betweenabout 100 and 400 rads, whole body irradiation to deplete or partiallydeplete the bone marrow of the recipient.

In preferred embodiments, the method further includes the administrationof thymic irradiation to the recipient, e.g., 300 to 700 rads of thymicirradiation.

Other preferred embodiments include: the step of prior to hematopoieticstem cell transplantation, depleting natural antibodies from the bloodof the recipient mammal, e.g., by hemoperfusing an organ, e.g., a liveror a kidney, obtained from a mammal of the second species. (In organhemoperfusion antibodies in the blood bind to antigens on the cellsurfaces of the organ and are thus removed from the blood.)

Other preferred embodiments further include the step of introducing intothe recipient a graft obtained from the donor, e.g., a graft which isobtained from a different organ than the hematopoietic stem cells, e.g.,a liver or a kidney.

In preferred embodiments the stem cells are introduced into therecipient prior to or simultaneous with transplantation of the graft.

In another aspect, the invention features a method of promoting, in arecipient mammal, preferably a primate, e.g., a human, acceptance of agraft obtained from a donor of the same species. The method includes:introducing, e.g., by intravenous injection into the recipient,hematopoietic stem cells, e.g. bone marrow cells or fetal liver orspleen cells, of a mammal, preferably the donor (preferably thehematopoietic stem cells home to a site in the recipient); (optionally)inactivating T cells of the recipient, e.g., by, prior to introducingthe hematopoietic stem cells into the recipient, introducing into therecipient an antibody capable of binding to T cells of the recipient;and, preferably, administering to the recipient a short course of animmunosuppressive agent, e.g., a short course of cyclosporine treatment,sufficient to inactivate recipient T cells, preferably thymic or lymphnode T cells. (Thymic or lymph node T cells might otherwise inhibit theengraftment or survival of the engineered cells.)

In preferred embodiments, the duration of the short course ofimmunosuppressive agent is: approximately equal to 30 days;approximately equal to or less than 8-12 days, preferably about 10 days;approximately equal to or less than two, three, four, five, or ten timesthe 8-12 or 10 day period mentioned above.

In preferred embodiments: the short course is begun before or at aboutthe time stem cells are introduced into the recipient; the short coursebegins on the day the stem cells are introduced into the recipient; theshort course begins within 1, 2, 4, 6, 8, or 10 days before or after thestem cells are introduced into the recipient.

In other preferred embodiments: the short course of an immunosuppressiveagent is administered in conjunction with one or both of an anti-T cellantibody, or thymic irradiation, e.g., 700 rads of thymic irradiation;the short course of immunosuppressive is sufficient to inactivate Tcells, e.g., thymic or lymph node T cells, which would not beinactivated by antibody-based inactivation of T cells, e.g.,inactivation by intravenous administrations of ATG antibodypreparations.

In preferred embodiments, the anti-T cell or NK cell antibody is anantihuman thymocyte polyclonal anti-serum; and the anti-serum isobtained from a horse or pig.

Other preferred embodiments include: the further step of, prior tohematopoietic stem cell transplantation, inactivating recipient NKcells, e.g., by introducing into the recipient mammal an antibodycapable of binding to NK cells of the recipient mammal; and those inwhich the same individual is the donor of both the graft and the bonemarrow.

Other preferred embodiments include: the step of, prior to hematopoieticstem cell transplantation, creating hematopoietic space in the recipientso as to promote engraftment and survival of the implanted stem cells,e.g., by irradiating the recipient mammal with low dose, e.g., betweenabout 100 and 400 rads, whole body irradiation to deplete or partiallydeplete the bone marrow of the recipient.

In preferred embodiments the method further includes administeringthymic irradiation to the recipient, e.g., 700 rads of thymicirradiation.

Other preferred embodiments include: the further step of, prior to bonemarrow transplantation, adsorbing natural antibodies from the blood ofthe recipient by hemoperfusing an organ, e.g., the liver, or a kidney,obtained from the donor.

Preferred embodiments include: the step of introducing into therecipient mammal, donor species specific stromal tissue, preferablyhematopoietic stromal tissue, e.g., fetal liver or thymus.

Other preferred embodiments further include the step of introducing intothe recipient, a graft obtained from the donor, e.g., a graft which isobtained from a different organ than the hematopoietic stem cells, e.g.,a liver or a kidney.

In preferred embodiments, the stem cells are introduced into therecipient prior to or simultaneous with transplantation of the graft.

Methods of inactivating T cells, preferably thymic or lymph node Tcells, of the invention can be used with yet other methods of inducingtolerance in which the inactivation of thymic or lymph node T cells isdesirable. For example, anti-thymic or lymph node T cell methods of theinvention can be used with: methods which use the implantation of axenogeneic thymic graft to induce tolerance, e.g., the methods describedin U.S. Ser. No. 08/163, 912 filed on Dec. 7, 1993; methods ofincreasing the level of the activity of a tolerance promoting or GVHDinhibiting cytokine or decreasing the level of activity of a toleranceinhibiting or GVHD promoting cytokine, e.g., the methods described inU.S. Ser. No. 08/114,072, filed Aug. 30, 1993; methods of using cordblood cells to induce tolerance, e.g., the methods described in U.S.Ser. No. 08/150,739; and the methods for inducing tolerance disclosed inSykes and Sachs, PCT/US94/01616, filed Feb. 14, 1994.

“Thymus-function deficient”, as used herein, refers to a condition inwhich the ability of an individual's thymus to support the maturation ofT cells is impaired as compared with a normal individual. Thymusdeficient conditions include those in which the thymus or thymusfunction is essentially absent.

“Tolerance”, as used herein, refers to the inhibition of a graftrecipient's ability to mount an immune response, e.g., to a donorantigen, which would otherwise occur, e.g., in response to theintroduction of a non self MHC antigen into the recipient. Tolerance caninvolve humoral, cellular, or both humoral and cellular responses. Theconcept of tolerance includes both complete and partial tolerance. Inother words, as used herein, tolerance include any degree of inhibitionof a graft recipient's ability to mount an immune response, e.g., to adonor antigen.

“A discordant species combination”, as used herein, refers to twospecies in which hyperacute rejection occurs when vascular organs aregrafted. Generally, discordant species are from different orders, whilenon-discordant species are from the same order. For example, rats andmice are non-discordant species, i.e. their MHC antigens aresubstantially similar, and they are members of the same order, rodentia.

“Hematopoietic stem cell”, as used herein, refers to a cell that iscapable of developing into mature myeloid and/or lymphoid cells.Preferably, a hematopoietic stem cell is capable of the long-termrepopulation of the myeloid and/or lymphoid lineages. Stem cells derivedfrom the cord blood of the recipient or the donor can be used in methodsof the invention. See U.S. Pat. No. 5,192,553, hereby incorporated byreference, and U.S. Pat. No. 5,004,681, hereby incorporated byreference.

“Miniature swine”, as used herein, refers to completely or partiallyinbred miniature swine.

“Graft”, as used herein, refers to a body part, organ, tissue, cells, orportions thereof

“Stromal tissue”, as used herein, refers to the supporting tissue ormatrix of an organ, as distinguished from its functional elements orparenchyma.

Restoring, inducing, or promoting immunocompetence, as used herein,means one or both of: (1) increasing the number of mature functional Tcells in the recipient (over what would be seen in the absence oftreatment with a method of the invention) by either or both, increasingthe number of recipient-mature functional T cells or by providing maturefunctional donor-T cells, which have matured in the recipient; or (2)improving the immune-responsiveness of the recipient, e.g., as ismeasured by the ability to mount a skin response to a recall antigen, orimproving the responsiveness of a of a T cell of the recipient, e.g., asmeasured by an in vitro test, e.g., by the improvement of aproliferative response to an antigen, e.g., the response to tetanusantigen or to an alloantigen.

A mature functional T cell, as used herein, is a T cell (of recipient ordonor origin) which responds to microbial antigens and tolerant torecipient and donor tissue.

“Lymph node or thymic T cell”, as used herein, refers to T cells whichare resistant to inactivation by traditional methods of T cellinactivation, e.g., inactivation by a single intravenous administrationof anti-T cell antibodies, e.g., anti-bodies, e.g., ATG preparation.

Restoring or inducing the thymus-dependent ability for T cellprogenitors to mature into mature T cells, as used herein, means eitheror both, increasing the number of functional mature T cells of recipientorigin in a recipient, or providing mature functional donor T cells to arecipient, by providing donor thymic tissue in which T cells can mature.The increase can be partial, e.g., an increase which does not bring thelevel of mature functional T cells up to a level which results in anessentially normal immune response or partial, e.g., an increase whichfalls short of bringing the recipient's level of mature functional Tcells up to a level which results in an essentially normal immuneresponse.

Methods of the invention will allow the induction of immunocompetence inpatients suffering from an immunodeficiency, e.g., a T cell deficiency,e.g., a thymic based immunodeficiency, e.g., a congenitalimmunodeficiency due to thymic aplasia or dysfunction, an acquiredimmune disorder, e.g., AIDS, immunoincompetence resulting form aneoplastic disease, or immunoincompetence resulting from a medicalprocedure, e.g., chemotherapy or radiation treatment. An acquired immunedeficiency is one which is due primarily to other than genetic defects.

At risk for AIDS, as used herein, refers to being HIV positive or havingAIDS.

“An immunosuppressive agent capable of inactivating thymic or lymph nodeT cells”, as used herein, is an agent, e.g., a chemical agent, e.g., adrug, which, when administered at an appropriate dosage, results in theinactivation of thymic or lymph node T cells. Examples of such agentsare cyclosporine, FK-506, and rapamycin. Anti-T cell antibodies, becausethey are comparatively less effective at inactivating thymic or lymphnode T cells, are not preferred for use as agents. An agent should beadministered in sufficient dose to result in significant inactivation ofthymic or lymph node T cells which are not inactivated by administrationof an anti-T cell antibody, e.g., an anti-ATG preparation. Putativeagents, and useful concentrations thereof, can be prescreened by invitro or in vivo tests, e.g., by administering the putative agent to atest animal, removing a sample of thymus or lymph node tissue, andtesting for the presence of active T cells in an in vitro or in vivoassay. Such prescreened putative agents can then be further tested intransplant assays.

“Short course of a immunosuppressive agent”, as used herein, means atransitory non-chronic course of treatment. The treatment should beginbefore or at about the time the treatment to induce tolerance is begun,e.g., at about the time, xenogeneic, allogeneic, genetically engineeredsyngeneic, or genetically engineered autologous stem cells areintroduced into the recipient. e.g., the short course can begin on theday the treatment to induce tolerance is begun, e.g., on the day,xenogeneic, allogeneic, genetically engineered syngeneic, or geneticallyengineered autologous stem cells are introduced into the recipient orthe short course can begin within 1, 2, 4, 6, 8, or 10 days before orafter the treatment to induce tolerance is begun, e.g., within 1, 2, 4,6, 8, or 10 days before or after xenogeneic, allogeneic, geneticallyengineered syngeneic, or genetically engineered autologous stem cellsare introduced into the recipient. The short course can last for: aperiod equal to or less than about 8-12 days, preferably about 10 days,or a time which is approximately equal to or is less than two, three,four, five, or ten times the 8-12 or 10 day period. Optimally, the shortcourse lasts about 30 days. The dosage should be sufficient to maintaina blood level sufficient to inactivate thymic or lymph node T cells. Adosage of approximately 15 mg/kg/day has been found to be effective inprimates.

“Help reduction”, as used herein, means the reduction of T cell help bythe inhibition of the release of at least one cytokine, e.g., any ofIL-2, IL-4, IL-6, gamma interferon, or TNF, from T cells of therecipient at the time of the first exposure to an antigen to whichtolerance is desired. The inhibition induced in a recipient's T cellsecretion of a cytokine must be sufficient such that the recipient istolerized to an antigen which is administered during the reduction ofhelp. Although not being bound by theory, it is believed that the levelof reduction is one which substantially eliminates the initial burst ofIL-2 which accompanies the first recognition of a foreign antigen butwhich does not eliminate all mature T cells, which cells may beimportant in educating and producing tolerance.

“A help reducing agent”, as used herein, is an agent, e.g., animmunosuppressive drug, which results in the reduction of cytokinerelease. Examples of help reducing agents are cyclosporine, FK-506, andrapamycin. Anti-T cell antibodies, because they can eliminate T cells,are not preferred for use as help reducing agents. A help reducing agentmust be administered in sufficient dose to give the level of inhibitionof cytokine release which will result in tolerance. The help reducingagent should be administered in the absence of treatments which promotecytokine, e.g., IL-2, release. Putative agents help reducing agents canbe prescreened by in vitro or in vivo tests, e.g., by contacting theputative agent with T cells and determining the ability of the treated Tcells to release a cytokine, e.g., IL-2. The inhibition of cytokinerelease is indicative of the putative agent's efficacy as a helpreducing agent. Such prescreened putative agents can then be furthertested in a kidney transplant assay. In a kidney transplant assay aputative help reducing agent is tested for efficacy by administering theputative agent to a recipient monkey and then implanting a kidney from aclass II matched class I and minor antigen mismatched donor monkey intothe recipient. Tolerance to the donor kidney (as indicated by prolongedacceptance of the graft) is indicative that the putative agent is, atthe dosage tested, a help reducing agent.

“Short course of a help reducing agent”, as used herein, means atransitory non-chronic course of treatment. The treatment should beginbefore or at about the time of transplantation of the graft.Alternatively, the treatment can begin before or at about the time ofthe recipient's first exposure to donor antigens. Optimally, thetreatment lasts for a time which is approximately equal to or less thanthe period required for mature T cells of the recipient species toinitiate rejection of an antigen after first being stimulated by theantigen. The duration of the treatment can be extended to a timeapproximately equal to or less than two, three, four, five, or tentimes, the period required for a mature T cell of the recipient speciesto initiate rejection of an antigen after first being stimulated by theantigen. The duration will usually be at least equal to the timerequired for mature T cells of the recipient species to initiaterejection of an antigen after first being stimulated by the antigen. Inpigs and monkeys, about 12 days of treatment is sufficient. Experimentswith cyclosporine A (10 mg/kg) in pigs show that 6 days is notsufficient. Other experiments in monkeys show that IL-2 administered onday 8, 9, or 10 of cyclosporine A treatment will result in rejection ofthe transplanted tissue. Thus, 8, 9, or 10 days is probably notsufficient in pigs. In monkeys, a dose of 10 mg/kg cyclosporine with ablood level of about 500-1,000 ng/ml is sufficient to induce toleranceto class II matched class I and minor antigen mismatched kidneys. Thesame blood level, 500-1,000 ng/ml, is sufficient to induce tolerance inpigs. Long-term administration of 5 mg/kg prevents rejection (by longterm immune suppression) but does not result in tolerance.

The help suppressing methods of the invention avoid the undesirable sideeffects of long-term or chronic administration of the broad spectrumimmune suppressants often used in transplantation. Long-term or chronicadministration of drugs such as Prednisone, Imuran, CyA, and, mostrecently FK506, have all had an important impact on the field oftransplantation. However, all of these drugs cause nonspecificsuppression of the immune system which must be titrated sufficiently toavoid rejection while not completely eliminating immune function.Patients who must stay on chronic immunosuppressive therapy for theremainder of their lives face major complications arising from too muchor too little immunosuppression, causing infection and rejection,respectively. The help suppressing methods of the invention are based onthe administration of a transitory short term high dose course of a helpreducing treatment.

“MHC antigen”, as used herein, refers to a protein product of one ormore MHC genes; the term includes fragments or analogs of products ofMHC genes which can evoke an immune response in a recipient organism.Examples of MHC antigens include the products (and fragments or analogsthereof) of the human MHC genes, i.e., the HLA genes. MHC antigens inswine, e.g., miniature swine, include the products (and fragments andanalogs thereof) of the SLA genes, e.g., the DRB gene.

Recipient thymic or lymph node T cells are responsible for significantresistance to implanted grafts, e.g., to transplanted hematopoieticcells or transplanted organs. It has been found that the usual methodsof T cell depletion or inactivation, e.g., the administration of anti-Tcell antibodies, often fall short of an optimum level of T celldepletion or inactivation. In particular, such methods fail to provideoptimum levels of depletion or inactivation of thymic or lymph node Tcells. Methods of the invention in which a short course of animmunosuppressant, e.g., cyclosporine, capable of inactivating recipientthymic or lymph node T cells is administered to the recipient, result inmore thorough inactivation of thymic or lymph node T cells and thus inimproved acceptance of graft tissue.

The retroviral methods of the invention allow the reconstitution of agraft recipient's bone marrow with transgenic autologous bone marrowcells expressing allogeneic or xenogeneic MHC genes. Expression of thetransgenic MHC genes confers tolerance to grafts which exhibit theproducts of these or closely related MHC genes. Thus, these methodsprovide for the induction of specific transplantation tolerance bysomatic transfer of MHC genes. Retroviral methods of the invention avoidthe undesirable side effects of broad spectrum immune suppressants whichare often used in transplantation.

Tolerance to transplantation antigens can be achieved through inductionof lymphohematopoietic chimerism by bone marrow transplantation (BMT).BMT across MHC barriers presents two major risks: if mature T cells arenot removed from the marrow inoculum the recipient may develop severegraft versus host disease (GVHD); removal of these cells often leads tofailure of engraftment. Retroviral methods of the invention, whichinduce specific tolerance by reconstitution of the recipient's bonemarrow with autologous (as opposed to allogeneic or heterologous) bonemarrow cells, allow tolerance to be conferred with minimal risk of GVHDand with minimal need to remove T cells from the marrow inoculum.

Retroviral methods of the invention can be combined with the helpsuppression and T cell inactivation methods of the invention to prolonggraft acceptance.

Hematopoietic cell transplant methods of the invention avoid theundesirable side effects of broad spectrum immune suppressants which areoften used in transplantation. Hematopoietic cell transplant methods ofthe invention can be combined with the help suppression and T cellelimination methods of the invention to prolong graft acceptance.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph showing growth of fetal pig THY/LIV graft vs. timeafter transplantation in the presence of mature mouse T cells in theperiphery.

FIG. 1B is a dot plot analysis of live peripheral white blood cells of arepresentative animal 16 weeks post-transplant. Upper left quadrant0.5%; upper right quadrant 12.1%.

FIG. 2 is a graph of mouse anti-pig mixed lymphocyte reactions (MLR's)performed to determine whether or not mouse T cells which matured in pigthymus grafts were tolerant to pig antigens.

FIG. 3 is a diagram of the GS4.5 retroviral construct.

FIG. 4 is a diagram of the GS4.5 proviral genome and the expectedtranscripts.

FIGS. 5 a and 5 b are representations of flow cytometry profile oftransduced cells.

FIG. 6 is a diagram of the transduction assay.

FIG. 7 is a diagram of genetic maps of the C57BL/10, B10.AKM, andB10.MBR strains.

FIG. 8 is a diagram of the FACS profile of spleen cells from a recipientof transduced bone marrow.

FIGS. 9 a and 9 b are graphs of survival versus time in skin graftexperiments.

FIGS. 10 a-d are diagrams of FACS analysis of thymocytes from graftrejecters, and controls.

FIG. 11 is a diagram of the N2-B19-H2b vector.

DETAILED DESCRIPTION

Maturation of Host T Cells in a Xenogeneic Thymus and Induction ofTolerance to a Xenograft by Xenogeneic Thymic Tissue

The following procedure was designed to promote the acceptance of axenograft thymus by a host and thusly to either or both: 1, lengthen thetime an implanted organ (a xenograft) survives in a xenogeneic hostprior to rejection; and 2, provide xenogeneic thymic tissue in whichhost T cells can mature.

In the case of an organ transplant, the organ can be any organ, e.g., aliver, e.g., a kidney, e.g., a heart. The two main strategies areelimination of natural antibodies and transplantation of thymic tissueto induce tolerance.

Preparation of the recipient for either organ transplantation or thymusreplacement includes any or all of the following steps. Preferably theyare carried out in the following sequence.

First, a preparation of horse anti-human thymocyte globulin (ATG) isintravenously injected into the recipient. The antibody preparationeliminates mature T cells and natural killer cells. If not eliminated,mature T cells might promote rejection of both the thymic transplantand, after sensitization, the xenograft organ. The ATG preparation alsoeliminates natural killer (NK) cells. NK cells probably have no effecton an implanted organ, but might act immediately to reject the newlyintroduced thymic tissue. Anti-human ATG obtained from any mammalianhost can also be used, e.g., ATG produced in pigs, although thus farpreparations of pig ATG have been of lower titer than horse-derived ATG.ATG is superior to anti-NK monoclonal antibodies, as the latter aregenerally not lytic to all host NK cells, while the polyclonal mixturein ATG is capable of lysing all host NK cells. Anti-NK monoclonalantibodies can, however, be used. In a relatively severelyimmunocompromised individual this step may not be necessary. As host (ordonor) T cells mature in the xenogeneic thymus they will be tolerant ofthe xenogeneic thymic tissue. Alternatively, as the host immune systemis progressively restored, it may be desirable to treat the host toinduce tolerance to the xenogeneic thymic tissue.

Optimally, the recipient can be thymectomized. In thymectomizedrecipients, recipient T cells do not have an opportunity todifferentiate in the recipient thymus, but must differentiate in thedonor thymus. In some cases it may be necessary to splenectomize therecipient in order to avoid anemia.

Second, the recipient can be administered low dose radiation. Althoughthis step is thought to be beneficial in bone marrow transplantation (bycreating hematopoietic space for newly injected bone marrow cells), itis of less importance in thymic grafts which are not accompanied by bonemarrow transplantation. However, a sublethal dose e.g., a dose aboutequal to 100, or more than 100 and less than about 400, rads, whole bodyradiation, plus 700 rads of local thymic radiation, can be used.

Third, natural antibodies can be adsorbed from the recipient's blood.(This is of more importance in organ grafts but can be used in thymusreplacement procedures as well.) Antibody removal can be accomplished byexposing the recipient's blood to donor or donor species antigens, e.g.,by hemoperfusion of a liver of the donor species to adsorbrecipient-natural antibodies. Pre-formed natural antibodies (nAb) arethe primary agents of graft rejection. Natural antibodies bind toxenogeneic endothelial cells and are primarily of the IgM class. Theseantibodies are independent of any known previous exposure to antigens ofthe xenogeneic donor. B cells that produce these natural antibodies tendto be T cell-independent, and are normally tolerized to self antigen byexposure to these antigens during development. The mechanism by whichnewly developing B cells are tolerized is unknown. The liver is a moreeffective adsorber of natural antibodies than the kidney. Again, thisstep may not be required, at least initially, in a relatively severelyimmunocompromised patient.

Donor thymic tissue, preferably fetal or neonatal thymic tissue isimplanted in the recipient. Fetal or neonatal liver or spleen tissue canbe included.

While any of these procedures may aid the survival of implanted thymictissue or another xenogeneic organ, best results are achieved when allsteps are used in combination.

Methods of the invention can be used to confer tolerance to allogeneicgrafts, e.g., wherein both the graft donor and the recipient are humans,and to xenogeneic grafts, e.g., wherein the graft donor is a nonhumananimal, e.g., a swine, e.g., a miniature swine, and the graft recipientis a primate, e.g., a human.

The donor of the implant and the individual that supplies thetolerance-inducing thymic graft should be the same individual or shouldbe as closely related as possible. For example, it is preferable toderive implant tissue from a colony of donors that is highly orcompletely inbred. The donor of the organ used for perfusion need not beclosely related to the donor of the implant or thymic tissue.

Xenograft Thymic Tissue Transplantation: Detailed Protocol

Immunocompetent C57BL/10 (B10) mice were used to test the ability of pigthymus to induce specific tolerance to discordant pig antigens. B10 micewere treated with a non-myeloablative conditioning regimen which haspreviously shown to permit induction of tolerance to rat xeno-antigensin mice, see e.g., Sharabi et al., 1990, J. Exp. Med. 172:195-202.Euthymic or thymectomized (ATX) mice received depleting doses of anti-Tcell and anti-NK cell mAbs, 7 Gy mediastinal irradiation and 3 Gy wholebody irradiation (WBI), and then received fetal swine thymus/liver(THY/LIV) transplants under the kidney capsule followed byadministration of 10⁸ fetal liver cells (FLC) i.p. Mice either receivedno further anti-T cell and anti-NK cell mAb treatments after 0 to 6weeks post-tx, or were maintained on chronic mAb treatment for theduration of the experiment.

Swine THY/LIV grafts grew initially in treated euthymic mice, butstopped growing after T cell and natural killer (NK) cell-depletingmonoclonal antibodies (mAbs) were discontinued, and these mice developedanti-pig IgG response. When euthymic mice were maintained on chronic mAbtreatment, the grafts enlarged markedly and no anti-pig IgG response wasobserved. Pig thymopoiesis was supported for at least 32 weeks duringchronic mAb administration, although no pig T cells were detected in theperiphery by flow cytometry (FCM). Percentages of intra-graft CD4⁺/CD8⁻,CD4⁻/CD8⁺, CD4⁺/CD8⁺, and CD4⁻/CD8⁻ pig thymocyte subsets were similarto those in normal pig thymus.

In contrast, swine THY/LIV grafts grew markedly in adult thymectomizedmice (ATX-THY/LIV) which received only a short (less than 6 weeks)course of mAb treatment post-transplant (post-tx). FCM analysis ofperipheral WBC in these mice 6 weeks after discontinuing mAb treatmentrevealed the presence of mature (αβ-TCR^(hi)) mouse T cells. Unlike Tcells in euthymic grafted mice, these cells were tolerant to pigantigens, as evidenced by the growth of swine THY/LIV grafts, (FIG. 1),and the absence of anti-pig IgG antibody responses. The majority (morethan 90%) of the αβ-TCR^(hi) T cells were CD4⁺/CD8⁻. FMC analyses 13 to26 weeks post-tx demonstrated normal mouse thymocyte subsets in swinethymi. For example, 7.9% CD4⁺/CD8⁻, 2.9% CD4⁻/CD8⁺, 85.5% CD4⁺/CD8⁺,3.7% CD4⁻/CD8⁻ and 11.6% αβ-TCR^(hi) thymocytes were found in a swineTHY/LIV graft by FCM 17 weeks post-tx compared to 3.5% CD4⁺/CD8⁻, 3.2%CD4⁻/CD8⁺, 87.8% CD4⁺/CD8⁺, 5.5% CD4⁻/CD8⁻ and 10.0% αβ-TCR^(hi)thymocytes in a normal B10 thymus. Fetal swine liver grafted without athymus fragment did not grow in control nAb-treated ATX-B10 mice andαβ-TCR^(hi) T cells did not appear in the periphery. Thus, the pigthymus was required for the development of mature mouse T cells.

Mouse anti-pig mixed lymphocyte reactions (MLR's) were performed todetermine whether or not mouse T cells which matured in pig thymusgrafts were tolerant to pig antigens. ATX-THY/LIV B10 mice (H-2^(b))mounted no anti-B10 or anti-pig responses, but demonstrated normalallo-responses against a fully MHC-mismatched allogeneic stimulator,B10.BR (H-2^(k)) (FIG. 2).

In order to determine if host bone marrow-derived cells wereparticipating in negative selection of the developing mouse thymocytes,fetal pig THY/LIV grafts were transplanted into both I-E⁺ (BALB/c nude)and I-E⁻ (ATX B10) recipients. I-E⁺ mice delete V_(β)11 T cells becauseof presentation in the thymus of an endogenous superantigen inassociation with I-E, whereas I-E⁻ mice do not delete this T cellfamily. The percentages of V_(β)11 T cells were therefore comparedbetween I-E⁺ and I-E⁻ recipients of fetal pig thymus grafts in whichmurine T cells developed in pig thymi. ATX B10 recipients were treatedas described above. BALB/c nude mice were depleted of NK cells andirradiated with 3 Gy WBI prior to transplant. These mice also developedlarge numbers of mature CD4⁺ T cells that migrated to the periphery.Complete deletion of V_(β)11 T cells was observed in the periphery ofBALB/c nude recipients of fetal swine thymus grafts (Table I),indicating that mouse I-E also participated in negative selection ofmouse T cells developing in pig thymi. Negative selection is most likelycarried out by murine Ia⁺dendritic cells which were detectedpredominantly in the cortico-medullary junction of swine thymus graftsby immunoperoxidase staining. In the ATX B10 recipients of swine THY/LIVgrafts, reduction in the percentage of V_(β)11 T cells was observedcompared to normal B10 mice (mean 2.8% of T cells±0.8 S.D., normal B105.2%, p<0.005) suggesting that the pig SLA DR class II, which sharessignificant homology with mouse I-E class II, may participate innegative selection of mouse T cells developing in the pig thymus graft(Table I). TABLE I N Strain Thy/Liv Graft % V_(β)8.1/8.2 % V_(β)11 4Normal C57BL/10 − 16.3 ± 2.2 5.2 ± 0.5 4 Normal BALB/c − 20.8 ± 0.3 0.2± 0.1 4 C57BL/10 + 16.7 ± 3.0 2.8 ± 0.8 5 BALB/c nude + 20.0 ± 3.7 0.4 ±0.3Table I. Clonal deletion in mice transplanted with swine THY/LIV grafts.B10 recipients were treated as described below. Normal percentages of Tcells staining with V_(β)8.1/8.2 demonstrates normal positive selectionin grafted mice of a V_(β)T cell family which is not deleted in I-E⁺ orI-E⁻ mice. BALB/c nude mice were depleted of NK cells using rabbitanti-Asialo-Gm1 serum, and were given 3 Gy WBI, and fetal swine THY/LIVgrafts implants under the kidney capsule, followed by injection of 10⁸FLC i.p. on Day 0. ACK-lysed splenocytes or peripheral white blood cells(red blood cells were removed by hypotonic shock) were collected 13 to19 weeks post-tx and analyzed by FCM for V_(β)11 T cell deletion. MurineF_(c)R's were blocked using rat anti-mouse F_(c)R mAb, 2.4G2, and thencells were stained with either fluoresceinated hamster anti-mouseV_(β)8.1/8.2 TCR (Pharmingen) or rat anti-mouse V_(β)11 TCR (Pharmingen)(green fluorescence) followed by phycoerythrin-conjugated rat anti-mouseCD4 and CD8 mAbs (Pharmingen) (orange fluorescence) and analyzed by twocolor FCM as described below. Fluoresceinated murine mAb HOPCI, with noknown reactivity to mouse cells, or rat anti-mouse IgG1 (ZymedLaboratories, Inc.) was used as the negative control mAb in the greenfluorescence. Phycoerythrin-conjugated mAb Leu4 (Becton-Dickinson), wasused as the negative control mAb in the orange fluorescence.Approximately 5,000 gated CD4⁺ and CD8⁺ cells were usually collected foranalysis of V_(β) families. Non-viable cells were excluded using thevital nucleic acid stain, propidium iodide. Percentages of positivecells were determined as described below. Results are presented as themean±SD of results obtained for individual mice. p value<0.005 for %V_(β)11 in normal B10 mice compared to THY/LIV-grafted B10 mice. p valueis >0.20 for % V_(β)11 in normal BALB/c mnice compared toTHY/LIV-grafted BALB/c nude mice.

These studies demonstrate that discordant xenogeneic thymic stroma iscapable of supporting mouse thymopoiesis and that CD4⁺/CD8⁻/αβ-TCR^(hi)T cells which are released into the periphery are phenotypically normal,functional and tolerant to donor xeno-antigens, and to host antigens.The lack of CD4⁻/CD8⁺/αβ-TCR^(hi) repopulation in the periphery may bedue to failure of mouse CD8 to interact with pig MHC class I molecules,as has been demonstrated for mouse anti-human responses, therebypreventing positive selection of CD8⁺ thymocytes by swine thymicepithelium. Since human CD8⁺ T cells are able to interact with pig MHCclass I directly, human CD8⁺ T cells should mature effectively in swinefetal thymus grafts.

Presumably, tolerance to pig antigens is not induced in euthymic micewhich receive swine THY/LIV grafts because mouse T cell progenitorsmature in the host thymus, which lacks the pig cells necessary totolerize developing mouse thymocytes. The non-myeloablative conditioningregimen used in this study permits engraftment of rat marrow andinduction of donor-specific tolerance in murine recipients. Tolerance isthought to be induced in this model by rat dendritic cells detected atthe cortico-medullary junction of the thymus of chimeric animals. In thepresent study, failure of pig hematopoietic stem cells, present in theFLC suspension administered on Day 0, to migrate to the mouse thymus maybe due to failure of homing and differentiation, possibly reflectingspecies specificity of cytokines and adhesion molecules. In ATXrecipients, on the other hand, mouse T cell progenitors home to the pigthymus graft and are tolerized pig antigens. No mouse TE is present inATX hosts, but mouse dendritic cells are detectable in THY/LIV graftsand probably mediate the observed clonal deletion of cells reactive tohost antigen. Although the decreased percentage of V_(β)11 T cells inATX B10 recipients of swine THY/LIV grafts suggests clonal deletion byswine cells, it is possible that there is a defect in the positiveselection of this V_(β) family on swine thymic stroma. However, thenormal percentages of V_(β)8.1/8.2 T cells in both ATX B10 and BALB/cnude recipients of swine THY/LIV grafts compared to those in normal B10and BALB/c mice suggests that no defect in positive selection ispresent. The observed tolerance could be explained if the swine thymicstroma either clonally deletes or anergizes developing mouse thymocytesreactive to donor xeno-antigens.

Survival of swine THY/LIV grafts in euthymic and thymectomized B10 micedepleted of T cells and NK cells, was determined as follows. 6-12 weekold euthymic or ATX C57BL/10 (B10) mice received i.p. injections of mAbsGK1.5 (anti-mouse CD4), 2.43 (anti-mouse CD8), 30-H12 (anti-mouseThy1.2) and PK136 (anti-mouse NK1.1) in depleting doses, as described inSharabi et al., on days −6 and −1 prior to transplantation. On eitherday −1 or day 0, 7 Gy localized thymic irradiation and 3 Gy whole bodyirradiation were administered to recipients, and second trimester(gestational day 36-72) fetal thymic and liver fragments, approximately1 mm³ in size, were transplanted under the kidney capsule via a midlinelaparotomy incision. (Thymic irradiation was not found to be necessaryfor mouse T cells to mature in pig thymus grafts in subsequentexperiments, and was therefore eliminated from the conditioningregimen.) After the abdomen was closed in two layers, 10⁸ fetal livercells (FLC) in suspension were injected i.p. Recipients were treated ona weekly basis post-tx with depleting doses of the same four mAbs for aperiod of 0-6 weeks. No difference in murine CD4⁺ T cell reconstitutionor tolerance to pig antigens was observed in mice which were treatedwith no mAb post-tx compared to those which received 6 weeks mAbtreatment post-tx. Some groups of control mice were maintained onchronic mAb treatment until the time of sacrifice.

As described above, an increase in fetal pig THY/LIV graft size wasobserved upon exploratory laparotomy performed at 5 and 19 weekspost-tx, despite the presence of mature CD4⁺/αβTCR^(hi) T cells in theperipheral blood (shown 16 weeks after mAbs were discontinued). Growthof fetal pig THY/LIV graft in the presence of mature mouse T cells inthe periphery was studied as follows. Peripheral WBC contained 12.1%CD4⁺/αβ-TCR^(hi) T cells and 0.5% CD8⁺/αβ-TRC⁺ T cells. Control ATX micewhich received fetal swine liver grafts without a thymus fragment didnot maintain their grafts, and developed less than 5% αβ-TRC⁺ T cells inthe periphery. ATX mice were conditioned as described above. Exploratorylaparotomies were performed at 5-6 and 15-19 weeks post-transplant tomeasure graft size. Mice were tail bled at regular intervals post-tx toobtain peripheral WBC which were prepared by hypotonic shock to removered blood cells. Cells were stained with a fluoresceinated ratanti-mouse CD4 mAb (Pharmigen) (green fluorescence) versus biotinylatedhamster anti-mouse αβTRC mAb (Pharmigen) plus phycoerythrin streptavidin(orange fluorescence) and analyzed by two-color flow cytometry (FCM)using either a FACScan or FACSort flow cytometer (Becton-Dickinson).Murine mAb HOPC1, with no known reactivity to pig or mouse cells, wasused as the negative control mAb in both the green and orangefluorescence. Percentages of positive cells were determined bysubtracting the percentage of cells staining with the control mAb HOPC1from the percentage of cells staining with the anti-mouse mAbs. A dotplot analysis of live peripheral white blood cells of a representativeanimal 16 weeks post-tx (16 weeks after mAbs were discontinued) is shownin FIG. 2. Overall, 57% (27/47) of ATX mice treated with this protocolmaintained swine grafts and reconstituted their CD4⁺ T cell compartment.In recent experiments this result was achieved in 90% (9/10) of micetreated with this regimen.

As described above, ATX-THY/LIV B10 (H-2^(b)) mice demonstrated specificunresponsiveness to pig antigens while maintaining normalallo-responsiveness to a fully MHC-mismatched B10.BR (H-2^(k))stimulator. Specific unresponsiveness of B10 mice transplanted withfetal pig THY/LIV grafts to pig antigens in mixed lymphocyte reaction(MLR) was determined as follows. Control ATX-B10 mice which received aswine liver graft without a thymus fragment (ATX-LIV) mounted noresponses to any stimulator, demonstrating the importance of the pigthymus graft in the development of functional mouse T cells. Positivecontrol anti-pig MLR was from a mouse immunized with a swine skin graft,since mice do not mount primary anti-pig responses. Sterile splenocytesuspensions from normal B10 (right diagonal bar), normal B10 graftedwith GG (SLA-I^(c)/SLA-II^(d)) pig skin 12 weeks earlier (GG′-B10 solidbar), normal B10BR (crosshatched bar), and thymectomized B10 miceconditioned with the non-myeloablative regimen described above andtransplanted with either a fetal pig (SLA-I^(d)/SLA-II^(d)) THY/LIVgraft (ATX-THY/LIV stippled bar), or a fetal pig liver graft only(ATX-LIV left diagonal bar) were ACK-lysed, washed and reconstituted inRPMI medium supplemented with 15% CPSR-2 (controlled processed serumreplacement, Sigma), 4% nutrient mixture (L-glutamine, nonessentialamino acids, sodium pyruvate and penicillin/streptomycin), 1% HEPESbuffer and 10⁻⁵M 2-me. Swine PBL were prepared by centrifugation over aFicoll-Hypaque layer. 4×10⁵ responders were incubated with either 4×10⁵murine stimulators (3 Gy) or 1×10⁵ swine stimulators (3 Gy) in a totalvolume of 0.2 ml of media at 37 □C for 4 days in 5% CO₂. Cultures werepulsed with 1 μCi ³H on the third day, harvested on the fourth day witha Tomtec automated harvester and counted on a Pharmacia LKB liquidscintillation counter. MLR's for all mice tested (N=3) were set up induplicate and pulsed on Days 3 and 4 and harvested on Days 4 and 5 withsimilar results.

Thus, if murine T cells are permitted to develop in a mouse thymus, theyare not tolerized to pig antigens, and they reject pig thymus/livergrafts. (Thus, if the recipient has significant thymic functionthymectomy is indicated.) If mouse T cells are continually depleted bymAb, swine thymopoiesis occurs in the swine thymus/liver graft. If amouse is thymectomized but not chronically treated with anti-T cellmAb's, mouse thymopoiesis occurs in the pig thymus, and these cells aretolerized to pig antigens.

Host T Cells which Mature in a Xenogeneic Thymus are Functional

B10.BR (full MHC mismatch to B10) and C3H.SW (minor antigen mismatchonly) skin grafts (1 mouse) were rejected by mouse T cells which hadmatured in a pig thymic graft, thus demonstrating their immunocompetenceand ability to recognize minor antigens in a host MHC-restrictedfashion. The grafts were full thickness tail skin grafts on the upperthorax with a skin bridge separating them. These results show that swinefetal thymic tissue can be used clinically to induce a state of specificxenograft tolerance while ensuring immunocompetence in thymectomizedrecipients.

Alternative Preparative Regimens

As is stated above, the depletion of NK cells, whole body irradiation,and thymic irradiation, can in some cases be dispensed with. As is shownby the experiments summarized below, inactivation or depletion of CD4⁺cells, e.g., by the administration of an anti-CD4 antibody, issufficient to allow growth of xenogeneic thymic tissue and maturation ofhost T cells in the xenogeneic thymic tissue. (The antibodies needed maydiffer depending on the species combination. E.g., in the case of ahuman recipient and a pig donor, because human CD8 will likely interactwith pig class I molecules, it may also be necessary to administeranti-CD8 antibodies.)

As is shown by the data in Table II below, graft growth and host T celldevelopment (as measured by the presence of peripheral T cells 9 and 10weeks post THY/LIV transplant) was seen in ATX mice treated withanti-CD4 antibodies and whole body irradiation. B10 mice received 3 Gywhole body irradiation and fetal pig THY/LIV graft and 10⁸ fetal livercells in experiments essentially similar to those described in theprevious section except that anti-CD4, anti-THY1.2, and anti NK cellantibodies were not administered. TABLE II THY/LIV graft growth and Tcell maturation does not require extensive antibody treatment. GRAFT %CD4⁺/% CD8⁺ Animal # TREATMENT L/R T CELLS (WBC) 639/40 NO ATX/αCD4/8/17.7/4.6  THY1.2/NK1.1 643/44 NO ATX/αCD4/8/ 9.4/2.6 THY1.2/NK1.1 601/02ATX +/ +/++ 11.1/0.8  αCD4/8/THY1.2/NK1.1 603/04 ATX +/ +/+ 1.7/1.2αCD4/8/THY1.2/NK1.1 605/06 ATX +/ −/− 0.6/0.3 αCD4/8/THY1.2/NK1.1 607/08ATX +/ ++/++ 0.9/0.6 αCD4/8/THY1.2/NK1.1 609/10 ATX +/ ++/+ 1.8/0.5αCD4/8/THY1.2/NK1.1 611/12 ATX +/ ++/++ 3.4/1.6 αCD4/8/THY1.2/NK1.1615/16 ATX + αCD4/8 +/++ 8.9/0.7 617/18 ″ +/− 2.1/0.3 619/20 ″ ++/++20.2/1.5  621/22 ″ +/− 1.1/0.1 623/24 ″ ++/++ 6.8/0.3 625/26 ATX + αCD4++/− 7.2/4.0 627/28 ″ −/+ 1.0/5.1 629/30 ″ ++/++ 10.1/3.7  631/32 ″ ++/+3.7/3.5 633/34 ″ −/− 0.4/3.7 635/36 ″ ++/++ 28.0/4.2 ATX = thymectomy;++ = large, bulky graft with vascularization;+ = moderate sized graft;− = poor graft (thin, poor tissue);αCD4/8/THY1.2/NK1.1 indicates the administration of the describedantibody.

As is shown by the data in Table III below, graft growth was seen in ATXmice treated with monoclonal antibodies but given no irradiation. Inthese experiments, B10 mice were given anti-CD4, CD8, THY1.2, and NK1.1monoclonal antibodies. TABLE III THY/LIV graft growth does not requirehost irradiation. GRAFT Animal # TREATMENT L/R 560/61 ATX + mAb's + 3 GyWBI ++/++ 562/63 ″ +/++ 564/65 ″ −/− 566/67 ″ −/+ 574/75 ″ −/− 576/77 ″−/− 578/79 ″ −/− 582/83 ″ +/− 552/53 ATX + mAb's (no WBI) +/− 554/55 ″++/− 556/57 ″ ++/+ 568/69 ″ ++/++ 570/71 ″ ++/++

As is shown by the data in Table IV below, host T cell development (asmeasured by the presence of peripheral T cells 7 weeks post THY/LIVtransplant) was seen in ATX mice given no irradiation and treated onlywith anti-CD4 antibodies. TABLE IV Rapid T cell recovery in THY/LIVgraft recipients with thymectomy and anti CD4 antibodies alone. Mean %(SD) peripheral blood Number of T cells at 7 weeks animals inpost-transplant group TREATMENT CD4⁺ CD8⁺ 3 NO ATX 12.9 (1.7) 2.7 (0.1)αCD4/8/THY1.2/NK1.1 + 3 Gy WBI 8 ATX  3.4 (2.4) 0.7 (0.5)αCD4/8/THY1.2/NK1.1 + 3 Gy WBI 7 ATX 19.5 (3.4) 7.1 (1.8) αCD4/Xenogeneic Thymic Tissue and Stem Cell Transplantation

The following procedure introduces donor thymic tissue and donor stemcells into the recipient and thus can be used to restore or induceimmune function or to lengthen the time an implanted organ (a xenograft)survives in a xenogeneic host prior to rejection.

In the case of an organ graft, the organ can be any organ, e.g., aliver, e.g., a kidney, e.g., a heart. The two main strategies areelimination of natural antibodies by organ perfusion, andtransplantation of tolerance-inducing bone marrow.

Preparation of the recipient includes any or all of the following steps.Preferably they are carried out in the following sequence.

Elimination of NK and T cells. First, a preparation of horse anti-humanthymocyte globulin (ATG) is intravenously injected into the recipient.The antibody preparation eliminates mature T cells and natural killercells. If not eliminated, mature T cells would promote rejection of boththe bone marrow transplant and, after sensitization, the xenograftitself of equal importance, the ATG preparation also eliminates naturalkiller (NK) cells. NK cells probably have no effect on the implantedorgan, but would act immediately to reject the newly introduced bonemarrow. Anti-human ATG obtained from any mama host can also be used,e.g., ATG produced in pigs, although thus far preparations of pig ATGhave been of lower titer than horse-derived ATG. ATG is superior toanti-NK monoclonal antibodies, as the latter are generally not lytic toall host NK cells, while the polyclonal mixture in ATG is capable oflysing all host NK cells. Anti-NK monoclonal antibodies can, however, beused.

Thymic tissue transplant. In cases where the procedure is to restore orinduce immunocompetence donor thymic tissue (preferably fetal orneonatal thymic tissue) is implanted in the recipient so that donor Tcells (and recipient T cells if the are present and functional) canmature. Fetal or neonatal liver or spleen tissue can be implanted withthe thymic tissue.

The presence of donor antigen in the thymus during the time when host Tcells are regenerating post-transplant is critical for tolerizing host Tcells. If donor hematopoietic stem cells are not able to becomeestablished in the host thymus and induce tolerance before host T cellsregenerate repeated doses of anti-recipient T cell antibodies may benecessary throughout the non-myeloablative regimen. Continuous depletionof host T cells may be required for several weeks. Alternatively, e.g.,if this approach is not successful, and tolerance (as measured by donorskin graft acceptance, specific cellular hyporesponsiveness in vitro,and humoral tolerance) is not induced in these animals, the approach canbe modified to include host thymectomy. In thymectomized recipients,host T cells do not have an opportunity to differentiate in a hostthymus, but must differentiate in the donor thymus. Immunocompetence canbe measured by the ability to reject a non-donor type allogeneic donorskin graft, and to survive in a pathogen-containing environment.

It may also be necessary or desirable to splenectomize the recipient inorder to avoid anemia.

Creation of hematopoietic space. The recipient is administered low doseradiation in order to make room for newly injected bone marrow cells. Asublethal dose e.g., a dose about equal to 100, or more than 100 andless than about 400, rads, whole body radiation, plus 700 rads of localthymic radiation, has been found effective for this purpose.

Natural antibody elimination. Natural antibodies are adsorbed from therecipient's blood by hemoperfusion of a liver of the donor species.Pre-formed natural antibodies-(nAb) are the primary agents of graftrejection. Natural antibodies bind to xenogeneic endothelial cells andare primarily of the IgM class. These antibodies are independent of anyknown previous exposure to antigens of the xenogeneic donor. B cellsthat produce these natural antibodies tend to be T cell-independent, andare normally tolerized to self antigen by exposure to these antigensduring development. The mechanism by which newly developing B cells aretolerized is unknown. The liver is a more effective adsorber of naturalantibodies than the kidney.

Implantation of donor stromal tissue. The next step in thenon-myeloablative procedure is to implant donor stromal tissue,preferably obtained from fetal liver, thymus, and/or fetal spleen, intothe recipient, preferably in the kidney capsule. Stem cell engraftmentand hematopoiesis across disparate species barriers is enhanced byproviding a hematopoietic stromal environment from the donor species.The stromal matrix supplies species-specific factors that are requiredfor interactions between hematopoietic cells and their stromalenvironment, such as hematopoietic growth factors, adhesion molecules,and their ligands.

Each organ includes an organ specific stromal matrix that can supportdifferentiation of the respective undifferentiated stem cells implantedinto the host. Although adult thymus may be used, fetal tissue obtainedsufficiently early in gestation is preferred because it is free frommature T lymphocytes which can cause GVHD. Fetal tissues also tend tosurvive better than adult tissues when transplanted. As an addedprecaution against GVHD, thymic stromal tissue can be irradiated priorto transplantation, e.g., irradiated at 1000 rads. As an alternative oran adjunct to implantation, fetal liver cells can be administered influid suspension.

Finally, bone marrow cells (BMC), or another source of hematopoieticstem cells, e.g., a fetal liver suspension, or cord blood stem cells, ofthe donor are injected into the recipient. Donor stem cells home toappropriate sites of the recipient and grow contiguously with remaininghost cells and proliferate, forming a chimeric lymphohematopoieticpopulation. By this process, newly forming B cells (and the antibodiesthey produce) are exposed to donor antigens, so that the transplant willbe recognized as self. Tolerance to the donor is also observed at the Tcell level in animals in which hematopoietic stem cell, e.g., BMC,engraftment has been achieved. When an organ graft is placed in such arecipient several months after bone marrow chimerism has been induced,natural antibody against the donor will have disappeared, and the graftshould be accepted by both the humoral and the cellular arms of theimmune system. This approach has the added advantage of permitting organtransplantation to be performed sufficiently long following transplantof hematopoietic cells, e.g., BMT, e.g., a fetal liver suspension, thatnormal health and immunocompetence will have been restored at the timeof organ transplantation. The use of xenogeneic donors allows thepossibility of using bone marrow cells and organs from the same animal,or from genetically matched animals. As liver is the major site ofhematopoiesis in the fetus, fetal liver can also serve as an alternativeto bone marrow as a source of hematopoietic stem cells.

While any of these procedures may aid the survival of an implantedorgan, best results are achieved when all steps are used in combination.Methods of the invention can be used to confer tolerance to allogeneicgrafts, e.g., wherein both the graft donor and the recipient are humans,and to xenogeneic grafts, e.g., wherein the graft donor is a nonhumananimal, e.g., a swine, e.g., a miniature swine, and the graft recipientis a primate, e.g., a human.

In the case of xenogeneic grafts, the donor of the implant and theindividual that supplies either the tolerance-inducing hematopoieticcells or the liver to be perfused should be the same individual orshould be as closely related as possible. For example, it is preferableto derive implant tissue from a colony of donors that is highly orcompletely inbred.

Detailed Protocol

In the following protocol for preparing a cynomolgus monkey for receiptof a kidney from a miniature swine donor, zero time is defined as themoment that the arterial and venous cannulas of the recipient areconnected to the liver to be perfused.

On day −1 a commercial preparation (Upjohn) of horse anti-humananti-thymocyte globulin (ATG) is injected into the recipient. ATGeliminates mature T cells and natural killer cells that would otherwisecause rejection of the bone marrow cells used to induce tolerance. Therecipient is anesthetized, an IV catheter is inserted into therecipient, and 6 ml of heparinized whole blood are removed beforeinjection. The ATG preparation is then injected (50 mg/kg)intravenously. Six ml samples of heparinized whole blood are drawn fortesting at time points of 30 min., 24 hrs and 48 hrs. Blood samples areanalyzed for the effect of antibody treatment on natural killer cellactivity (testing on K562 targets) and by FACS analysis for lymphocytesubpopulations, including CD4, CD8, CD3, CD11b, and CD16. Preliminarydata from both assays indicate that both groups of cells are eliminatedby the administration of ATG. If mature T cells and NK cells are noteliminated, ATG can be re-administered at later times in the procedure,both before and after organ transplantation.

Sublethal irradiation is administered to the recipient between days −1and −8. Irradiation is necessary to eliminate enough of the recipient'sendogenous BMC to stimulate hematopoiesis of the newly introducedforeign BMC. Sublethal total body irradiation is sufficient to permitengraftment with minimal toxic effects to the recipient. Whole bodyradiation (150 Rads) was administered to cynomolgus monkey recipientsfrom a bilateral (TRBC) cobalt teletherapy unit at 10 Rads/min. Localirradiation of the thymus (700 Rads) was also employed in order tofacilitate engraftment.

Natural antibodies are a primary cause of organ rejection. To removenatural antibodies from the recipient's circulation prior totransplantation, on day 0 an operative adsorption of natural antibodies(nAB) is performed, using a miniature swine liver, as follows. At −90minutes the swine donor is anesthetized, and the liver prepared forremoval by standard operative procedures. At −60 minutes the recipientmonkey is anesthetized. A peripheral IV catheter is inserted, and a 6 mlsample of whole blood is drawn. Through mid-line incision, the abdominalaorta and the vena cava are isolated. Silastic cannulas containing sideports for blood sampling are inserted into the blood vessels.

At −30 minutes the liver is perfused in situ until it turns pale, andthen removed from the swine donor and placed into cold Ringers Lactate.The liver is kept cold until just prior to reperfusion in the monkey. Aliver biopsy is taken. At −10 minutes the liver is perfused with warmalbumin solution until the liver is warm (37 degrees).

At 0 time the arterial and venous cannulas of the recipient areconnected to the portal vein and vena cava of the donor liver andperfusion is begun. Liver biopsies are taken at 30 minutes and 60minutes, respectively. Samples of recipient blood are also drawn forserum at 30 minutes and 60 minutes respectively. At 60 minutes the liveris disconnected from the cannulas and the recipient's large bloodvessels are repaired. The liver, having served its function of adsorbingharmful natural antibodies from the recipient monkey, is discarded.Additional blood samples for serum are drawn from the recipient at 2,24, and 48 hours. When this procedure was performed on two sequentialperfusions of swine livers, the second liver showed no evidence of mildischemic changes during perfusion. At the end of a 30 minute perfusionthe second liver looked grossly normal and appeared to be functioning,as evidenced by a darkening of the venous outflow blood compared to thearterial inflow blood in the two adjacent cannulas. Tissue sections fromthe livers were normal, but immunofluorescent stains showed IgM onendothelial cells. Serum samples showed a decrease in naturalantibodies.

To promote long-term survival of the implanted organ through T-cell andB-cell mediated tolerance, donor bone marrow cells are administered tothe recipient to form chimeric bone marrow. The presence of donorantigens in the bone marrow allows newly developing B cells, and newlysensitized T cells, to recognize antigens of the donor as self, andthereby induces tolerance for the implanted organ from the donor. Tostabilize the donor BMC, donor stromal tissue, in the form of tissueslices of fetal liver, thymus, and/or fetal spleen are transplantedunder the kidney capsule of the recipient. Stromal tissue is preferablyimplanted simultaneously with, or prior to, administration ofhematopoietic stem cells, e.g., BMC, or a fetal liver cell suspension.

To follow chimerism, two color flow cytometry can be used. This assayuses monoclonal antibodies to distinguish between donor class I majorhistocompatibility antigens and leukocyte common antigens versusrecipient class I major histocompatibility antigens.

BMC can in turn be injected either simultaneously with, or preceding,organ transplant. Bone marrow is harvested and injected intravenously(7.5×10⁸/kg) as previously described (Pennington et al., 1988,Transplantation 45:21-26). Should natural antibodies be found to recurbefore tolerance is induced, and should these antibodies cause damage tothe graft, the protocol can be modified to permit sufficient timefollowing BMT for humoral tolerance to be established prior to organgrafting.

The approaches described above are designed to synergistically preventthe problem of transplant rejection. When a kidney is implanted into acynomolgus monkey following liver adsorption of natural antibodies,without use of bone marrow transplantation to induce tolerance, renalfunctions continued for 1-2 days before rejection of the kidney. Whenfour steps of the procedure were performed (adsorption of naturalantibodies by liver perfusion, administration of ATG, sublethalirradiation and bone marrow infusion, followed by implant of a porcinekidney into a primate recipient), the kidney survived 7 days beforerejection. Despite rejection of the transplanted organ, the recipientremained healthy.

When swine fetal liver and thymic stromal tissue were implanted underthe kidney capsule of two sublethally irradiated SCID mice, 25-50% ofperipheral blood leukocytes were of donor lineage two weekspost-transplantation. A significant degree of chimerism was not detectedin a third animal receiving fetal liver without thymus. These proceduresdid not employ any chemical immunosuppressants.

Other Embodiments

Other embodiments are within the following claims.

For example, implanted grafts may consist of organs such as liver,kidney, heart; body parts such as bone or skeletal matrix; tissue suchas skin, intestines, endocrine glands; or progenitor stem cells ofvarious types.

The methods of the invention may be employed with other mammalianrecipients (e.g., rhesus monkeys, humans) and may use other mammaliandonors (e.g., primates, swine, sheep, dogs).

The methods of the invention may be employed in combination, asdescribed, or in part.

The method of introducing bone marrow cells may be altered, particularlyby (1) increasing the time interval between injecting hematopoietic stemcells and implanting the graft; (2) increasing or decreasing the amountof hematopoietic stem cells injected; (3) varying the number ofhematopoietic stem cell injections; (4) varying the method of deliveryof hematopoietic stem cells; (5) varying the tissue source ofhematopoietic stem cells, e.g., a fetal liver cell suspension may beused; or (6) varying the donor source of hematopoietic stem cells.Although hematopoietic stem cells derived from the graft donor arepreferable, hematopoietic stem cells may be obtained from otherindividuals or species, or from genetically-engineered completely orpartially inbred donor strains, or from in vitro cell culture.

Methods of preparing the recipient for transplant of hematopoietic stemcells may be varied. For instance, the recipient may undergo asplenectomy or a thymectomy. The latter would preferably by administeredprior to the non-myeloablative regimen, e.g., at day −14.

Hemoperfusion of natural antibodies may: (1) make use of other vascularorgans, e.g., liver, kidney, intestines; (2) make use of multiplesequential organs; (3) make use of varying the length of time each organis perfused; (4) make use of varying the donor of the perfused organ.Irradiation of the recipient may make use of: (1) of varying theadsorbed dose of whole body radiation below the sublethal range; (2) oftargeting different body parts (e.g., thymus, spleen); (3) varying therate of irradiation (e.g., 10 rads/min, 15 rads/min); or (4) varying thetime interval between irradiation and transplant of hematopoietic stemcells; any time interval between 1 and 14 days can be used, and certainadvantages may flow from use of a time interval of 4-7 days. Antibodiesintroduced prior to hematopoietic cell transplant may be varied by: (1)using monoclonal antibodies to T cell subsets or NK cells (e.g.,anti-NKH1_(A), as described by U.S. Pat. No. 4,772,552 to Hercend, etal., hereby incorporated by reference); (2) preparing anti-human ATG inother mammalian hosts (e.g., monkey, pig, rabbit, dog); or (3) usinganti-monkey ATG prepared in any of the above mentioned hosts.

As an alternative or adjunct to hemoperfusion, host antibodies can bedepleted by administration of an excess of hematopoietic cells.

Stromal tissue introduced prior to hematopoietic cell transplant, e.g.,BMT, may be varied by: (1) administering the fetal liver and thymustissue as a fluid cell suspension; (2) administering fetal liver orthymus stromal tissue but not both; (3) placing a stromal implant intoother encapsulated, well-vascularized sites; or (4) using adult thymusor fetal spleen as a source of stromal tissue.

As is discussed herein, it is often desirable to expose a graftrecipient to irradiation in order to promote the development of mixedchimerism. The inventor has discovered that it is possible to inducemixed chimerism with less radiation toxicity by fractionating theradiation dose, i.e., by delivering the radiation in two or moreexposures or sessions. Accordingly, in any method of the inventioncalling for the irradiation of a recipient, e.g., a primate, e.g., ahuman, recipient, of a xenograft or allograft, the radiation can eitherbe delivered in a single exposure, or more preferably, can befractionated into two or more exposures or sessions. The sum of thefractionated dosages is preferably equal, e.g., in rads or Gy, to theradiation dosage which can result in mixed chimerism when given in asingle exposure. The fractions are preferably approximately equal indosage. For example, a single dose of 700 rads can be replaced with,e.g., two fractions of 350 rads, or seven fractions of 100 rads.Hyperfractionation of the radiation dose can also be used in methods ofthe invention. The fractions can be delivered on the same day, or can beseparated by intervals of one, two, three, four, five, or more days.Whole body irradiation, thymic irradiation, or both, can befractionated.

Methods of the invention can include recipient splenectomy.

As is discussed herein, hemoperfusion, e.g., hemoperfusion with a donororgan, can be used to deplete the host of natural antibodies. Othermethods for depleting or otherwise inactivating natural antibodies canbe used with any of the methods described herein. For example, drugswhich deplete or inactivate natural antibodies, e.g., deoxyspergualin(DSG) (Bristol), or anti-IgM antibodies, can be administered to therecipient of an allograft or a xenograft. One or more of, DSG (orsimilar drugs), anti-IgM antibodies, and hemoperfusion, can be used todeplete or otherwise inactivate recipient natural antibodies in methodsof the invention. DSG at a concentration of 6 mg/kg/day, i.v., has beenfound useful in suppressing natural antibody function in pig tocynomolgus kidney transplants.

Some of the methods described herein use irradiation to createhematopoietic space, and thereby prepare a recipient for theadministration of allogeneic, xenogeneic, syngeneic, or geneticallyengineered autologous, stem cells. In any of the methods describedherein, particularly primate or clinical methods, it is preferable tocreate hematopoietic space for the administration of such cells bynon-lethal means, e.g., by administering sub-lethal doses ofirradiation, bone marrow depleting drugs, or antibodies. The use ofsublethal levels of bone marrow depletion allows the generation of mixedchimerism in the recipient. Mixed chimerism is generally preferable tototal or lethal ablation of the recipient bone marrow followed bycomplete reconstitution of the recipient with administered stem cells.

Xenogeneic thymic tissue is easier to obtain and in general is lesslikely to harbor human pathogens. Thus, xenogeneic thymic tissue ispreferred in methods for restoring or inducing immunocompetence.Allogeneic thymic tissue can however be used in these methods.

Some of the methods described herein include the administration ofthymic irradiation, e.g., to inactivate host thymic-T cells or tootherwise diminish the host's thymic-T cell mediated responses to donorantigens. It has been discovered that the thymic irradiation called forin allogeneic or xenogeneic methods of the invention can be supplementedwith, or replaced by, other treatments which diminish (e.g., bydepleting thymic-T cells and/or down modulating one or more of the Tcell receptor (TCR), CD4 co-receptor, or CD8 co-receptor) the host'sthymus function, e.g., the host's thymic-T cell mediated response. Forexample, thymic irradiation can be supplemented with, or replaced by,anti-T cell antibodies (e.g., anti-CD4 and/or anti-CD8 monoclonalantibodies) administered a sufficient number of times, in sufficientdosage, for a sufficient period of time, to diminish the host's thymic-Tcell mediated response.

For best results, anti-T cell antibodies should be administeredrepeatedly. E.g., anti-T cell antibodies can be administered one, two,three, or more times prior to donor thymus or bone marrowtransplantation. Typically, a pre-thymus or bone marrow transplantationdose of antibodies will be given to the patient about 5 days prior tothymus or bone marrow transplantation. Additional, earlier doses 6, 7,or 8 days prior to thymus or bone marrow transplantation can also begiven. It may be desirable to administer a first treatment then torepeat pre-thymus or bone marrow administrations every 1-5 days untilthe patient shows excess antibodies in the serum and about 99% depletionof peripheral T cells and then to perform the bone marrowtransplantation. Anti-T cell antibodies can also be administered one,two, three, or more times after thymus or donor bone marrowtransplantation. Typically, a post-thymus or bone marrow transplanttreatment will be given about 2-14 days after bone marrowtransplantation. The post thymus or bone marrow administration can berepeated as many times as needed. If more than one administration isgiven the administrations can be spaced about 1 week apart. Additionaldoses can be given if the patient appears to undergo early or unwanted Tcell recovery. Preferably, anti-T cell antibodies are administered atleast once (and preferably two, three, or more times) prior to donorthymus or bone marrow transplantation and at least once (and preferablytwo, three, or more times) after donor thymus or bone marrowtransplantation.

It has also been discovered that much or all of the preparative regimen,if called for, can be delivered or administered to a recipient, e.g., anallograft or xenograft recipient, within a few days, preferably within72, 48, or 24 hours, of transplantation of tolerizing stem cells and/orthe graft. This is particularly useful in the case of humans receivinggrafts from cadavers. Accordingly, in any of the methods of theinvention calling for the administration of treatments prior to thetransplant of stem cells and/or a graft, e.g., treatments to inactivateor deplete host antibodies, treatments to inactivate host T cells or NKcells, or irradiation, the treatment(s) can be administered, within afew days, preferably within 72, 48, or 24 hours, of transplantation ofthe stem cells and/or the graft. In particular, primate, e.g., human,recipients of allografts can be given any or all of treatments toinactivate or deplete host antibodies, treatments to inactivate host Tcells or NK cells, or irradiation, within a few days, preferably within72, 48, or 24 hours, of transplantation of stem cells and/or the graft.For example, treatment to deplete recipient T cells and/or NK cells,e.g., administration of ATG, can be given on day −2, −1, and 0, and WBI,thymic irradiation, and stem cell, e.g., bone marrow stem cells,administered on day 0. (The graft, e.g., a renal allograft, istransplanted on day 0).

As described in PCT/US94/01616, hereby incorporated by reference, it hasbeen discovered that there is a permissible time period (“window”) forhematopoietic stem cell engraftment following the creation of space(e.g., by whole body irradiation) for the donor hematopoietic stem cellsin a recipient. It has further been discovered that space created forhematopoietic stem cell engraftment can be monitored over time bymonitoring peripheral white blood cell levels in a recipient. Themyelosuppressive treatment sufficient to create hematopoietic spacegenerally results in a reduction in white blood cell (WBC) levels (asrevealed, e.g., by WBC counts) and the WBC reduction serves as a markerfor the presence of hematopoietic space. The marker is a conservativeone since WBC counts may recover at a time when space is still presentin an animal.

Accordingly, in any method which involves hematopoietic stem celltransplantation, and thus also requires the creation of hematopoieticspace in a recipient, transplantation can be performed during thepermissible window for engraftment following creation of space for thehematopoietic stem cells. Likewise, in any method in which space iscreated for exogenously administered hematopoietic stem cells, whiteblood cell levels can be followed to monitor space for the donorhematopoietic stem cells (i.e., to assess the permissible window forengraftment). Examples of procedures involving hematopoietic stem celltransplantation include: 1) conditioning of a recipient for an allo- orxenograft in which hematopoietic stem cell transplantation is performedin conjunction with transplantation of another allo- or xenograft; 2)treatment of various hematopoietic disorders, including leukemias,lymphomas and other hematopoietic malignancies and genetic hematopoieticdisorders (e.g., adenosine deaminase deficiency, bare lymphocytesyndrome and other congenital immunodeficiency diseases) in whichhematopoietic stem cell transplantation is performed therapeutically;and 3) transplantation of genetically modified hematopoietic stem cells(e.g., genetically modified autologous hematopoietic stem cells) todeliver a gene product to a recipient (e.g., as gene therapy).

Accordingly, methods of the invention include a method of determining ifa myelosuppressive or hematopoietic-space inducing treatment issufficient to create hematopoietic space. The method includesadministering a myelosuppressive treatment to a recipient, anddetermining the level of white blood cells in the recipient, e.g., bydetermining the WBC count of the recipient, a depression in the level ofwhite blood cells being indicative of the presence or induction ofhematopoietic space.

As is discussed in PCT/US94/01616, hereby incorporated by reference, andelsewhere herein, the engraftment of exogenously supplied hematopoieticstem cells can be promoted by treating the recipient of the cells so asto induce hematopoietic space in the recipient. Hematopoietic space iscommonly induced by radiation, but other procedures can replace orreduce the need for WBI. For example, space can be created by treatingthe recipient with a monoclonal antibody against MHC class I antigensexpressed by the recipient (see e.g., Voralia, M. et al. (1987)Transplantation 44:487) or space can be created by treating therecipient with myelosuppressive drugs (see e.g., Lapidot, T. et al.(1990) Proc. Natl. Acad. Sci. USA 87:4595).

It has also been found that the direct introduction of donor antigen,e.g., donor hematopoietic stem cells, into the thymus of a recipient,can modify the immune response of the recipient. Thus, embodiments ofthe invention include methods of promoting the acceptance a graft (e.g.,by prolonging the acceptance the graft) by a recipient, by introducinginto the recipient, donor antigen. The graft can be an allograft, e.g.,a graft from a primate e.g., a human, which is introduced into a primateof the same species. The graft can be a concordant or discordantxenograft. E.g., the graft can be a miniature swine graft introducedinto a second species, e.g., a primate, e.g., a human.

Induction of Tolerance

The invention provides several methods of inducing tolerance to foreignantigens, e.g., to antigens on allogeneic or xenogeneic tissue or organgrafts. These methods can be used individually or in combination. Forexample, it has been discovered that short-term administration of a helpreducing agent, e.g., a short high dose course of cyclosporine A (CsA)significantly prolongs graft acceptance. (Preferably the help reductionregimen of the invention substantially eliminates the initial burst ofIL-2 which accompanies the first recognition of an antigen but does noteliminate mature T cells. This is distinct from anti-T cell antibodytreatments which eliminate mature T cells.)

It has also been discovered that a short course of an immunosuppressant,e.g., cyclosporine, can be used to inactivate T cells which wouldotherwise promote the rejection of a graft.

Experiments which show the effect of cyclosporine-induced tolerance onrenal allografts in primates are described in section I below. The helpsuppression methods of the invention can be combined with other methodsfor prolonging graft acceptance. Section II below discusses implantationof retrovirally transformed bone marrow cells to induce tolerance to MHCdisparity. This method can be combined with help suppression methods ofthe invention, e.g., a short course of high dose of cyclosporine toinduce tolerance to class I and other minor disparities.

Section III below discusses implantation of bone marrow cells to inducetolerance to MHC disparity. This method can be combined with a shortcourse of high dose cyclosporine administration to induce tolerance toclass I and other minor disparities. A short course of cyclosporine, toeliminate T cells, can also be combined with bone marrow transplant.

I. A Short Post-Transplant Course of High Dose of Cyclosporine(Administered in the Absence of Agents which Stimulate Cytokine Release)Prolongs Acceptance of Partially Matched Allografts in Primates.

Renal transplants were performed between cynomolgus monkeys with orwithout a brief course of T cell-help-eliminating immunosuppression inthe form of a short course of high dose cyclosporine. This regimensignificantly prolonged acceptance of the grafts. Monkeys which receiveda post-transplant course of high dose cyclosporine (without Prednisone)were tolerant to kidney grafts from MLC (mixed lymphocyte culture assay)nonreactive class I and minor antigen mismatched donors for over 65days, see below. Monkeys which did not receive post-transplantcyclosporine rejected grafts of the same disparity in less than 20 days.This work is discussed in more detail below.

Animals. Cynomolgus monkeys were purchased from Charles River ResearchLaboratories. The animals were quarantined, tested for a full battery ofpathogens, and then housed in environmentally controlled rooms in strictconformance to the N.I.H. Guide for Care and use of Laboratory Animals,in an AAALAC accredited facility.

Typing. Animals were typed by a standard complement mediatedcytotoxicity assay for class I antigens and by MLC nonreactivity forclass II matching.

Immunosuppression. An intravenous preparation of CsA (Sandimmune, i.v.)was obtained from Sandoz Pharmaceuticals Corporation, Hanover, N.J.Monkeys received 12 doses of about 10 mg/kg with the first dose given 4hours prior to graft revascularization. The CsA was diluted in 250 ml ofnormal saline and infused intravenously over 1 hour. The CsA wasadministered without other immunosuppressants. The duration of therapywas 12 days. The suitable dosage in pigs is about 15 mg/kg deliveredintramuscularly. The dosage in either animal should be such that a bloodlevel of about 500-1,000 ng/ml is maintained.

Renal Function, Rejection, and Pathology. Renal function was followed bycreatinine and BUN levels in serum. Pathology was by biopsy. Biopsieswere performed at day 7, then weekly for 2 months, then monthly.

Results. Control recipients (no cyclosporine) rejected transplantedkidneys in less than 15 days. The results with 6 cyclosporine treatedanimals were as follows: animal 1, died on day 65 (i.e., 65 days aftertransplant), the transplanted was rejected, (it should be noted that theblood cyclosporine level of this animal was below 500 ng/ml during thefirst 7 days after transplant); animal 2, died on day 65 from bleedingfrom a biopsy, the transplanted kidney was normal at the time of death;animal 3 died on day 82, some rejection was apparent on day 55; animal 4was still normal at day 70 (at which time the experiment was still inprogress); animal 5 was still normal at day 40 (at which time theexperiment was still in progress); and animal 6 was still normal at day26 (at which time the experiment was still in progress).

II. A Short Course of High Dose Cyclosporine (Administered in theAbsence of Treatments which Stimulate the Release of Cytokines, e.g. theAbsence of Prednisone) to Induce Tolerance to Class I and Other MinorDisparities Combined with Implantation of Retrovirally Transformed BoneMarrow Cells to Induce Tolerance to Class II Disparity.

Retroviral Transformation

Retroviral transformation allows the reconstitution of a graftrecipient's bone marrow with transgenic bone marrow cells, preferablyautologous bone marrow cells, expressing allogeneic or xenogeneic MHCgenes. Expression of the transgenic MHC genes confers tolerance tografts which exhibit the products of these or closely related MHC genes.Thus, these methods provide for the induction of specifictransplantation tolerance by somatic transfer of MHC genes. Retroviralintroduction of MHC genes can be used alone or combined with the T cellhelp reducing methods described herein. This approach is discussed indetail below.

MHC Genes: MHC genes for a variety of species are well studied. Forexample the HLA genes in man, see, e.g., Hansen et al., 1989, The MajorHistocompatibility Complex, In Fundamental Immunology 2d ed., W. E.Paul, ed., Raven Press Ltd., New York, hereby incorporated by reference,and the SLA genes in swine, see e.g., Sachs et al., 1988, Immunogenetics28:22-29, hereby incorporated by reference, have been cloned andcharacterized.

A gene encoding a MHC antigen can be used in methods of the invention toconfer tolerance to a graft which displays that or a closely related MHCantigen. Methods of the invention can be used to confer tolerance toallogeneic grafts, e.g., wherein both the graft donor and the recipientare humans, and to xenogeneic grafts, e.g., wherein the graft donor is anonhuman animal, e.g., a swine, e.g., a miniature swine, and the graftrecipient is a human.

The individual which supplies the MHC genes should be as geneticallysimilar as possible, particularly in terms of the MHC genes, to theindividual which supplies the graft. For example, in allogeneic graftswherein the implant donor is a human and the implant recipient is ahuman it is preferable to use MHC genes from the donor. In thisembodiment, MHC probes, e.g., a probe from a third person or a syntheticconsensus probe, can be used to isolate DNA encoding the MHC gene orgenes of the implant donor individual. This allows the closest matchbetween the genes used to confer tolerance and the genes which expressMHC antigens on the graft.

In xenogeneic grafts, the implant donor individual and the individualwhich supplies the tolerance conferring DNA should be the sameindividual or should be as closely related as possible. For example, itis preferable to derive implant tissue from a colony of donors which ishighly inbred and, more preferably, which is homozygous for the MHCgenes. This allows the single cloned MHC sequence to be used for manygraft recipients.

Transformation of bone marrow cells: MHC genes can be introduced intobone marrow cells by any methods which allows expression of these genesat a level and for a period sufficient to confer tolerance. Thesemethods include e.g., transfection, electroporation, particle gunbombardment, and transduction by viral vectors, e.g., by retroviruses.

Recombinant retroviruses are a preferred delivery system. They have beendeveloped extensively over the past few years as vehicles for genetransfer, see e.g., Eglitis et al., 1988, Adv. Exp. Med. Biol. 241:19.The most straightforward retroviral vector construct is one in which thestructural genes of the virus are replaced by a single gene which isthen transcribed under the control of regulatory elements contained inthe viral long terminal repeat (LTR). A variety of single-gene-vectorbackbones have been used, including the Moloney murine leukemia virus(MoMuLV). Retroviral vectors which permit multiple insertions ofdifferent genes such as a gene for a selectable marker and a second geneof interest, under the control of an internal promoter can be derivedfrom this type of backbone, see e.g., Gilboa, 1988, Adv. Exp. Med. Biol.241:29.

The elements of the construction of vectors for the expression of aprotein product, e.g., the choice of promoters is known to those skilledin the art. The most efficient expression from retroviral vectors isobserved when “strong” promoters are used to control transcription, suchas the SV 40 promoter or LTR promoters, reviewed in Chang et al., 1989,Int. J. Cell Cloning 7:264. These promoters are constitutive and do notgenerally permit tissue-specific expression. However, in the case ofclass I genes, which are normally expressed in all tissues, ubiquitousexpression is acceptable for functional purposes. Housekeeping genepromoters, e.g., the thymidine kinase promoter, are appropriatepromoters for the expression of class II genes.

The use of efficient packaging cell lines can increase both theefficiency and the spectrum of infectivity of the produced recombinantvirions, see Miller, 1990, Human Gene Therapy 1:5. Murine retroviralvectors have been useful for transferring genes efficiently into murineembryonic, see e.g., Wagner et al., 1985, EMBO J. 4:663; Griedley etal., 1987 Trends Genet. 3:162, and hematopoietic stem cells, see e.g.,Lemischka et al., 1986, Cell 45:917-927; Dick et al., 1986, Trends inGenetics 2:165-170.

A recent improvement in retroviral technology which permits attainmentof much higher viral titers than were previously possible involvesamplification by consecutive transfer between ecotropic and amphotropicpackaging cell lines, the so-called “ping-pong” method, see e.g., Kozaket al., 1990, J. Virol. 64:3500-3508; Bodine et al., 1989, Prog. Clin.Biol. Res. 319: 589-600.

Transduction efficiencies can be enhanced by pre-selection of infectedmarrow prior to introduction into recipients, enriching for those bonemarrow cells expressing high levels of the selectable gene, see e.g.,Dick et al., 1985, Cell 42:71-79; Keller et al., 1985, Nature318:149-154. In addition, recent techniques for increasing viral titerspermit the use of virus-containing supernatants rather than directincubation with virus-producing cell lines to attain efficienttransduction, see e.g., Bodine et al., 1989, Prog. Clin. Biol. Res.319:589-600. Because replication of cellular DNA is required forintegration of retroviral vectors into the host genome, it may bedesirable to increase the frequency at which target stem cells which areactively cycling e.g., by inducing target cells to divide by treatmentin vitro with growth factors, see e.g., Lemischka et al., 1986, Cell45:917-927, a combination of IL-3 and IL-6 apparently being the mostefficacious, see e.g., Bodine et al., 1989, Proc. Natl. Acad. Sci.86:8897-8901, or to expose the recipient to 5-fluorouracil, see e.g.,Mori et al., 1984, Jpn. J. Clin. Oncol. 14 Suppl. 1:457-463, prior tomarrow harvest, see e.g., Lemischka et al., 1986, Cell 45:917-927; Changet al., 1989, Int. J. Cell Cloning 7:264-280.

N2A or other Moloney-based vectors are preferred retroviral vectors fortransducing human bone marrow cells.

Preparative Regimen For The Introduction of Transformed Bone MarrowCells To prepare for bone marrow cells the recipient must undergo anablation of the immune response which might otherwise resistengraftment.

The preparative regimens necessary to permit engraftment of modifiedautologous hematopoietic stem cells may be much less toxic than thoseneeded for allogeneic bone marrow transplantation—preferably requiringonly depletion of mature T cells with monoclonal antibodies, as has beenrecently demonstrated in a mouse model, see Sharabi et al., 1989, J.Exp. Med. 169:493-502. It is possible that transient expression may besufficient to induce tolerance, which may then be maintained by thetransplant even if expression on hematopoietic cells is lost, as hasbeen observed for heart transplants in a mixed xenogeneic bone marrowtransplant model, Ildstad et al., 1985, Transplant. Proc. 17: 535-538.

Graft and help reduction: The help reducing methods described above canbe administered in conjunction with transplantation of the graft, as isdescribed above.

Sustained Expression of a Swine Class II Gene in Murine Bone MarrowHematopoietic Cells by Retroviral-Mediated Gene Transfer

Overview: The efficacy of a gene transfer approach to the induction oftransplantation tolerance in miniature swine model was shown by usingdouble-copy retroviral vectors engineered to express a drug-resistancemarker (neomycin) and a swine class II DRB cDNA. Infectious particlescontaining these vectors were produced at a titer of >1×10⁶G418-resistant colony-forming units/ml using both ecotropic andamphotropic packaging cell lines. Flow cytometric analysis ofDRA-transfected murine fibroblasts subsequently transduced withvirus-containing supernatants demonstrated that the transferredsequences were sufficient to produce DR surface expression.Cocultivation of murine bone marrow with high-titer producer lines leadsto the transduction of 40% of granulocyte/macrophage colony-formingunits (CFU-GM) as determined by the frequency of colony formation underG418 selection. After nearly 5 weeks in long-term bone marrow culture,virus-exposed marrow still contained G418-resistant CFU-GM at afrequency of 25%. In addition, virtually all of the transduced andselected colonies contained DRB-specific transcripts. These results showthat a significant proportion of very primitive myelopoietic precursorcells can be transduced with the DRB recombinant vector and that vectorsequences are expressed in the differentiated progeny of these cells.These experiments are described in detail below.

Construction and Screening of SLA-DRB Recombinant Retroviruses As inman, Lee et al., 1982, Nature 299:750-752, Das et al., 1983, Proc. Natl.Acad. Sci. USA 80:3543-3547, the sequence of the swine DRA gene isminimally polymorphic. Therefore, transduction of allogeneic DRB cDNAsinto bone marrow cells should be sufficient to allow expression ofallogeneic class II DR molecules on cells committed to express thisantigen.

Details of retroviral constructs are given in FIG. 3. Two types ofretroviral constructs, GS4.4 and GS4.5, were prepared. The diagram inFIG. 3 depicts the GS4.5 retroviral construct. The arrows in FIG. 3indicate the directions of transcription. In GS4.5, the orientation ofDRB cDNA transcription is the same as viral transcription. In GS4.4 (notshown), the TK promoter and the DRB cDNA were inserted into the 3′ LTRof N2A in the reverse orientation of transcription with respect to viraltranscription and the simian virus 40 3′ RNA processing signal wasadded. pBSt refers to Bluescript vector sequence (Stratagene). Thethymidine kinase (TK) promoter was contained within the 215-base-pair(bp) Pvu II-Pst I fragment from the herpes simplex virus TK gene,McKnight, 1980 Nucleic Acids Res. 8:5949-5964. The simian virus 40 3′RNA processing signal was contained within the 142-bp Hpa I-Sma Ifragment from the pBLCAT3 plasmid, Luckow et al., (1987) Nucleic AcidsRes. 15:5490-5497, (see FIG. 3). Sequence analysis of the junctions ofthe promoter, the class II cDNA, and the vector sequences confirmed thatthe elements of the constructs were properly ligated.

These retroviral constructs were transfected into the amphotropicpackaging cell line PA317, and transfectants were selected inG418-containing medium. A total of 24 and 36 clones, transfected,respectively, with the GS4.4 and GS4.5 recombinant plasmids, were testedby PEG precipitation of culture supernatants and slot-blot analysis ofviral RNA. Of these, 8 and 12 clones were found, respectively, to bepositive for DRB, although the DRB signal was consistently weaker forthe GS4.4-derived clones. Analysis of genomic and spliced transcriptsfrom GS4.5 cells by dot-blot analysis of PEG-precipitated particlesrevealed heterogeneity among viral transcripts in various clonestransfected by GS4.5. In one experiment, two clones contained DRB⁺/Neo⁺viral RNA, two contained DRB⁺/Neo⁻ RNA, two contained DRB⁻/Neo⁺ RNA, andone showed no class II or Neo signal. G418-resistance (G418^(r)) titerdetermination of supernatants from DRB-positive clones confirmed thatthe average titer produced by GS4.5-transfected clones (10³-10⁴ CFU/ml)was significantly higher than that of the GS4.4-transfected clones(10²-10³ CFU/ml). Further transduction experiments were, therefore,conducted with the best clone, named GS4.5 C4, which produced an initialG418^(r) titer of 3×10⁴ CFU/ml.

Plasmid preparation, cloning procedures, DNA sequencing, RNApreparations, Northern blots, and RNA slot blots were performed bystandard methods, Sambrook et al., 1989, Molecular Cloning: A LaboratoryManual 2nd Ed. (Cold Spring Harbor Lab., Cold Spring Harbor). Finalwashes of blots were carried out in 0.1×SSPE (1×SSPE =0.18 M NaCl/10 mMsodium phosphate, pH 7.4/1 mM EDTA) at 60° C. for 30 min.

The packaging cell lines PA317, Miller et al., 1986, Mol. Cell. Biol.6:2895-2902, GP+E-86, Markowitz et al., 1988, J. Virol 62:1120-1124,psiCRIP, Danos et al., 1988, Proc. Natl. Acad. Sci. USA 85:6460-6464,and their derivatives were maintained at 37° C. in Dulbecco's modifiedEagle's medium (DMEM; GIBCO) with 10% (vol/vol) fetal bovine serum(CELLect Silver; Flow Laboratories) supplemented with 0.1 mMnonessential amino acids (Whittaker Bioproducts), antibiotics penicillin(5 units/ml), and streptomycin (5 μg/ml).

Improvement of the Viral Titer of the C4 Clone

Since recent data indicated that supernatants containing high retroviraltiters were the best candidates for transducing bone marrow cells,Bodine et al., 1990, Proc. Natl. Acad. Sci. USA 87:3738-3742, the titerof the C4 producer clone was increased by “ping-pong” amplification,Bestwick et al., 1988, Proc. Natl. Acad. Sci. USA 85:5404-5408.Supernatant from nearly confluent C4 cultures was used to transduceGP+E-86 ecotropic packaging cells and G418 selection was applied.Forty-eight clones were isolated and screened by PEG precipitation forproduction of viral particles. Supernatants from 18 of these clones wereDRB-positive by dot-blot analysis of viral RNA and had G418^(r) titersbetween 0.5 and 3.5×10⁴ CFU/ml). One positive clone was then amplifiedby the ping-pong technique with the amphotropic hygromycin-resistantpackaging line psiCRIP. Supernatants from 48 hygromycin-resistant cloneswere examined for presence of DRB-positive viral RNA by PEGprecipitation and their G418^(r) titers were determined. All the cloneswere positive by dot-blot analysis with the DRB probes and producedtiters between 1×10⁵ and 1×10⁷ CFU/ml. Amphotropic clone GS4.5 A4, whichproduced the highest titer, was tested for the presence of helper virusby the S+L-assay. No replication-competent helper virus was detected.

Amplification of virus titer was achieved by the ping-pong technique.Since there is evidence that psiCRIP packaging cells are less prone toproduce helper virus than PA317 when using certain types of vectors,Miller, 1990, Hum. Gene Therapy 1:5-14, DRB recombinant virions wereprepared using the psiCRIP/GP-E-86 producer combination. Titervalues>1×10⁷ CFU/ml with no detectable amphotropic helper viruses wereobtained, confirming that this strategy produced safe viral particlessuitable for in vivo experiments.

Northern blot analysis of GS4.5-producing clones C4, A9, and A4, eachderived from a different packaging cell line, showed a conservedhybridization pattern. RNA species corresponding to the full-lengthviral genome, the spliced Neo transcript, and the DRB transcription unitwere observed with additional RNA species. High molecular size speciesobserved in these experiments may constitute a read-through transcriptstarting from the TK promoter and ending in the other long terminalrepeat (LTR). In contrast to many of the virion-producer clones-obtainedby transfection that presented erratic DRB transcripts, those obtainedby transduction showed stable DRB hybridization patterns suggesting thatno recombination events occurred during the amplification procedure.

Retroviral titers were determined as follows. Replication-defectiveretroviral particles were produced from packaging cell lines initiallytransfected with recombinant construct using the standard calciumphosphate precipitation method, Wigler et al., 1978, Cell 14:725-733.Retrovirus production was estimated by the drug-resistance titer(G418-resistant colony-forming units/ml, CFU/ml) as described, Bodine etal., 1990, Proc. Natl. Acad. Sci. USA 87:3738-3742. Except for thepsiCRIP line, G418 (GIBCO) selection was carried out in active componentat 500 μg/ml for 10-12 days. Hygromycin B selection was applied topsiCRIP-derived packaging clones in medium containing active drug at 50μg/ml for 10 days. Replication-competent helper virus titer was assayedon PG4 feline cells by the S⁺L⁻ method, Bassen et al., 1971, Nature229:564-566.

PEG precipitation of viral particles was performed as follows. Virionscontained in 1 ml of culture supernatant were precipitated with 0.5 mlof 30% (wt/vol) polyethylene glycol (PEG) for 30 min. at 4° C. Aftercentrifugation, the pellets were treated with a mixture of RNaseinhibitors (vanadyl ribonuclease complex, BRL),phenol/chloroform-extracted, and ethanol-precipitated. Pellets were thenresuspended in 15.7% (vol/vol) formaldehyde and serial dilutions weredotted onto nitrocellulose membrane.

Analysis of DRB Transcription in Packaging Cell Clones To test foraccurate transcription of the introduced DRB cDNA within the differentproducer clones, Northern blots containing RNAs isolated from theseclones were hybridized with the DRB and Neo probes. FIG. 4 depicts thestructure of the provirus genome and the expected sizes of transcriptsinitiated from either the viral LTR or the TK promoters. Each of thethree GS4.5-containing clones, which were derived from PA317 (clone C4),GP+E-86 (clone A9), and psiCRIP (clone A4) cells, showed DRB-positivetranscripts. As reported, Hantzopoulos et al., 1989, Proc. Natl. Acad.Sci. USA 86:3519-3523, the unspliced genomic RNA (band a) and thespliced Neo transcript (band b) were observed. In addition a transcriptuniquely hybridizable with the DRB probe was detected that correspondsto the size predicted (1700 bases, band c) for the DRB cDNAtranscription unit.

Surface Expression of the SLA-DR Antigen on Transduced Fibroblasts An invitro assay was developed to examine surface expression of the SLA-DRantigen on murine fibroblasts. Flow cytometry (FCM) profiles shown inFIG. 5 demonstrate that G418^(r) titers of 3×10⁴ (clone C4) weresufficient to promote expression of the DR antigen on the cell surfaceof transduced DRA transfectants. In FIG. 5 solid lines indicate DR cellsurface expression (anti-DR antibody binding) (22% and 75% of the bulkpopulation of cells 3 days after transduction with GS4.5 C4, (B) andGS4.5 A4 (C), respectively); dashed lines indicate anti-mouse class Iantibody binding (positive control); dotted lines indicate anti-pig CD8antibody binding (negative control). Twenty-two percent of the bulkpopulation of transduced cells were DR-positive and subclones maintainedclass II expression for more than 5 months. The increase in titer (cloneA4) correlated with an increase in the number of cells transduced (75%of the transduced population was DR-positive) and with the brightness ofthe DR signal.

The class II transduction assay was performed as diagrammed in FIG. 6.NIH 3T3 cells were transfected with the SLA-DRA^(d) cDNA inserted in aplasmid expression vector, Okayama et al., 1982, Mol. Cell. Biol.2:161-170. Approximately 3×10⁴ cells of a stable DRA transfectant (clone11/12.2F) that expressed a high level of DRA mRNA were then transducedovernight with 1 ml of DRB-containing retroviral supernatant. Cells weresubsequently cultivated in fresh DMEM supplemented with 10% fetal bovineserum and antibiotics for 2 additional days and examined for cellsurface expression of the DR antigen by FCM analysis.

The class II transduction assay described here provides a fast andsimple method to test both the expression and functional titer ofretroviral constructs. By using cells transfected with DRA, the need forlengthy double selection after transduction by two separated vectors,Yang et al., 1987, Mol. Cell Biol. 1:3923-3928; Korman et al., 1987,Proc. Natl. Acad. Sci. USA 84:2150-2154, is obviated. Cell-surfaceexpression of DR heterodimers was demonstrated by FCM analysis 3 daysafter transduction, providing direct evidence that the transferredsequences were sufficient to produce significant level of DR β chain.More importantly, this test allows determination of “functional” titersbased on the expression of the gene of interest rather than on that ofthe independently regulated drug-resistance marker.

The SLA-DRB probe was an EcoRI cDNA fragment containing the completecoding sequence of the DR β chain, Gustafsson et al., 1990, Proc. Natl.Acad Sci. USA 87:9798-9802. The neomycin phosphotransferase gene (Neo)probe was the Bcl I-Xho I fragment of the N2A retroviral plasmid,Hantzopoulos et al., 1989, Proc. Natl. Acad Sci. USA 86:3519-3523.

Expression of Porcine DRB cDNA Transduced into Murine Bone MarrowProgenitor Cells The efficiency with which myeloid clonogenic precursorswere transduced was determined by assaying for CFU-GM with and without aselecting amount of G418 after exposure of bone marrow cells toGS4.5-derived virions. Comparison of the number of colonies that formedin the presence and absence of the drug, for two experiments, indicatedthat ≈40% of the initial population of myeloid progenitor cells weretransduced. The frequency of G418^(r) CFU-GM was again determined aftera sample of the transduced marrow was expanded under long-term cultureconditions for 33 days. Twenty-five percent of the progenitors presentafter 33 days in culture still gave rise to colonies under G418selection. Colonies of cells arisen from CFU-GM were examined for thepresence of DRB-specific transcripts by converting RNA into cDNA andthen performing PCR amplification as described herein and in Shafer etal., 1991 Proc. Natl. Acad. Sci. USA 88:9670. A 360-bp DRB-specificproduct was detected in five of six G418-selected colonies from freshlytransduced marrow, whereas all six colonies similarly derived fromtransduced progenitors present after 33 days in culture were positive.An additional band of 100 bp observed in some of the samples probablyreflects the stochastic nature of nonspecific priming events.DRB-specific transcripts were also detected in the bulk population ofdrug-resistant colonies and in producer cells but were not detected incontrols such as a bulk population of untransduced colonies, fibroblastsused to provide carrier RNA, and a bulk population of transducedcolonies processed as above but without reverse transcriptase. Theselatter data demonstrate that the PCR signal was dependent on thesynthesis of cDNA, excluding the possibility that provirus, rather thanviral message, was responsible for the amplified fragment.

Recent improvements including modifications of the virus design,increase of viral titers, use of growth factors to stimulate precursorcells, and selection of stem cells prior to transduction have been shownto improve long-term expression of transduced genes in the hematopoieticcompartment, Bodine et al., 1990, Proc. Natl. Acad. Sci. USA87:3738-3742; Bodine et al., 1989, Proc. Natl. Acad. Sci. USA86:8897-8901; Wilson et al., 1990, Proc. Natl. Acad. Sci. USA87:439-443; Kang et al., 1990, Proc. Natl. Acad. Sci. USA 87:9803-9807;Bender et al., 1989, Mol. Cell. Biol. 9:1426-1434. The experimentsherein show the applicability of the retroviral gene-transfer techniquein achieving expression of major histocompatibility complex class IIgenes transferred into hematopoietic cells. To determine the efficiencywith which developmentally primitive hematopoietic cells weretransduced, the frequency of G418^(r) CFU-GM was assessed afterexpanding infected marrow cells kept for 33 days in long-term cultures.Expression of the exogenous DRB cDNA was also monitored in cells derivedfrom transduced CFU-GM present either immediately after infection orafter an extended culture period. Virtually all of the coloniesindividually tested were positive for DRB-specific transcript,suggesting that the DRB recombinant vector is suitable for expression inmurine hematopoietic cells.

Bone marrow cells were obtained from the femora of 6- to 12-week-oldfemale C57BL/10 mice and were prepared as described, Ildstad et al.,1984, Nature 307:168-170. Methylcellulose colony assays forgranulocyte/macrophage colony-forming units (CFU-GM), Eaves et al.,1978, Blood 52:1196-1210, were performed as described using 5% (vol/vol)murine interleukin 3 culture supplement (Collaborative Research).Long-term Dexter-type bone marrow cultures were initiated in 60-mmculture dishes with 2×10⁷ nucleated cells, Eaves et al., 1987, CRC Crit.Rev. Oncol. Hematol. 7:125-138.

Bone marrow cells were transduced essentially as described, Bodine etal., 1989, Proc. Natl. Acad. Sci. USA 86:8897-8901. Briefly, bone marrowwas harvested for 6-12-week-old female C57BL/10 donors that had beentreated 2 days with 5-fluorouracil (150 mg/kg). Prestimulation wasperformed by incubating 1×10⁶ cells per ml for 2 days in long-termDexter-type bone marrow culture medium to which was added 7.5%interleukin 3 culture supplement and recombinant human interleukin 6(200 units/ml; gift from J. Jule, National Institutes of Health,Bethesda, Md.). Marrow cells were transduced for 48 hr by adding 5×10⁶cells per 10-cm plate containing nearly confluent virus-producers,Polybrene (8 mg/ml), and the cytokines described above.

Detection of DRB-Specific Transcripts in CFU-Derived Colonies wasperformed as follows. Cells corresponding to individual CFU colonies andto colonies present on an entire plate (bulk) were first extracted frommethylcellulose cultures by dilution in phosphate-buffered saline andcentrifugation. These cells were then combined with 1×10⁶ NIH 3T3 cells(to provide carrier RNA), and total RNA was prepared using the guanidineisothiocyanate/CsCl method. First-strand cDNA was prepared from 20 μg oftotal RNA using the Invitrogen Red Module kit. cDNA was then subjectedto 50 cycles of PCR amplification in the presence of the SLADRB-specific oligonucleotides 04 (5′-CCACAGGCCTGATCCCTAATGG) (Seq. I.D.No. 1) and 17 (5′-AGCATAGCAGGAGCCTTCTCATG) (Seq. I.D. No. 2) using theCetus GeneAmp kit as recommended (Perkin-Elmer/Cetus). Reaction productswere visualized after electrophoresis on a 3% NuSieve agarose gel (FMC)by staining with ethidium bromide.

FCM analysis was performed with a FAC-Scan II fluorescence-activatedcell sorter (Becton Dickenson) on cells stained with the anti-DRmonoclonal antibody 40D, Pierres et al., 1980, Eur. J. Immunol.10:950-957, an anti-H-2^(d) allo antiserum, or the anti-porcine CD8monoclonal antibody 76-2-11, Pescovitz et al., 1984, J. Exp. Med.160:1495-1505, followed by fluorescein isothiocyanate-labeled goatanti-mouse antibodies (Boehringer Mannheim).

Expression of Allogeneic Class II cDNA in Swine Bone Barrow CellsTransduced with a Recombinant Retrovirus

A MHC gene (DRB) was transferred into clonogenic progenitor cells fromswine using a recombinant retroviral vector (GS4.5) and a transductionprotocol designed to be applicable in vivo. Both the selectable drugresistance gene and the allogeneic class II cDNA transferred by thisvector were expressed in the progeny of these transduced progenitors.Expression of the Neo gene was monitored functionally by colonyformation under G418 selection, while the presence of class IItranscripts was detected by PCR analysis. With this latter method, thetranscriptional expression of both endogenous and virally derived DRBgenes in transduced and selected colonies were demonstrated.

Primary porcine fibroblasts were cultured with high titer viralsupernatants, and then analyzed by northern blotting using probesspecific for DRB and Neo. A specific transcript was observed which wasuniquely hybridizable with the DRB probe and migrated at the positionpredicted (1700 bases) for the DRB cDNA transcription unit arising fromthe TK promoter and terminating at the LTR 3′ RNA processing site.

To determine whether GS4.5 containing virions could transduce swinemyelopoietic progenitor cells a colony assay adapted for swine CFU-GMwas used. Transductions were carried out by incubating bone marrow froma donor of the SLAC haplotype in high titer viral supernatant.Comparisons of the number of colonies which formed in the presence andabsence of G418 for a total of 5 independent experiments indicated that5% to 14% of CFU-GM were transduced.

Colonies of cells originating from transduced CFU-GM were examined forthe presence of DRB-specific transcripts by converting RNA into cDNA,and then performing PCR amplification. Utilizing a polymorphic Sau3AIrestriction site absent from the endogenous DRB^(c) gene, the presenceof DRB^(d)-specific transcripts was unambiguously demonstrated. Gelelectrophoresis of the PCR product demonstrated that a 183/177 bpdoublet indicative of the vector-derived DRB^(d) transcript wasamplified in samples derived not only from pools of transduced andselected CFU-GM progeny, but also from at least 4 out of 6 individualcolonies tested. A 360 bp PCR fragment, indicative of endogenous DRB^(c)transcripts, was also amplified not only as expected from PBL isolatedfrom an SLAC donor, but also from both of the pooled colony samples anda number of the individual colony samples.

Construction of the retrovirus GS4.5, and production of high titer viralsupernatants was as described above. Detection of DRB-specifictranscripts in CFU-derived colonies by PCR of cDNA were described aboveand as follows. Bone marrow from an SLA^(c) donor was exposed toGS4.5-containing virions, and G418 selected colonies were tested for thepresence of DRB^(c) (endogenous) and DRB^(d) (vector derived) specifictranscripts by PCR of cDNA followed by digestion with Sau3AI and agarosegel electrophoresis. Controls were as follows: template synthesizedeither in the presence or absence of reverse transcriptase; templatederived from cells producing GS4.5-containing virions, from PBL isolatedfrom SLA^(c) or SLA^(d) donors, and from untransduced producer cellsused as carrier RNA.

Transduction of bone marrow was performed as follows. Swine bone marrowwas harvested as previously described (Pennington et al., 1988,Transplantation 45:21-26) and transductions were carried out byincubating marrow cells in high titer viral supernatants at an m.o.i. of3-5 in the presence of 8 μg of polybrene per ml at 37° C. for 5 hr.Myeloid progenitors were assayed by colony formation in methylcellulosecultures using PHA-stimulated swine lymphocyte conditioned medium as asource of growth factors. Selective medium contained 1.2 mg/ml activeG418.

Transduced bone marrow was administered to a lethally irradiatedminiature swine. At 5 weeks peripheral blood lymphocytes were analyzedby Southern, northern, and cell-surface FACS analyses. By all of thesetest there was evidence of presence of the transduced allogeneic classII gene in these cells and for expression of the product of this gene.In particular, northern analysis showed bands characteristic of thetranscribed cDNA, and FACS analysis with a combination of alloantiseraand monoclonal antibodies to DR showed presence of the transduced alleleof DR beta on the surface of peripheral lymphocytes.

Allogeneic Tolerance

Development of the B10.MBR-B10.AKM Strain Combination In an attempt tomaintain strains which are truly congenic for the MHC, a program ofcontinuous backcrossing of each congenic line to a common backgroundpartner strain was instituted more than 15 years ago. Backcross animalswere intercrossed and appropriate progeny selected by serologic typingin order to reestablish each congenic line. Thus, C57BL/10 was used asone reference background strain and all other congenic lines on the B10background were backcrossed once every six to ten generations to thisC57BL/10 line.

During the backcrossing of each congenic line to its pedigreed referenceline, there is of course the chance for an intra-MHC recombination eventto occur. Typing of the intercross (F2) generation serologically revealssuch recombinant events, and when the recombinant provides a newhaplotype of potential interest for genetic studies, it is outcrossedand then intercrossed to produce a homozygous new recombinant H-2haplotype. One of the most valuable of such recombinants originating inthis colony is the B10.MBR line, Sachs et al., 1979, J. Immunol.123:1965-1969, which was derived from a recombination event during thebackcrossing of B10.AKM to C57BL/10. Because this strain was the firstto separate K^(b) from I^(k) it has been used extensively in studies ofR-2 immunogenetics. In addition, in combination with the parentalB10.AKM strain, the B10.MBR offers the possibility of examining anisolated K gene as the only MHC difference between these two strains.Thus, as illustrated in FIG. 7, introduction of the K^(b) gene intoB10.AKM bone marrow stem cells, could theoretically lead to expressionof all cell surface MHC antigens of the B10.MBR. Expression on bonemarrow derived cell populations produces transplantation tolerance tothe product of the transduced gene, and this tolerance can be tested bya tissue graft from the B10.MBR strain.

Reconstitution of Myeloablated Mice with Transduced Bone Marrow Eightyprospective donor B10.AKM mice were treated with 150 mg/kg 5 FU on day−7. Bone marrow was harvested from these mice on day −5, treated withanti-CD4 and anti-CD8 monoclonal antibodies (mAbs) plus complement toremove mature T cells, and cultured for five days with N2-B19-H2bvirus-containing supernatant (H2) from the psi-Crip packaging cell line.As a control, one-half of the marrow was cultivated with supernatantfrom control packaging cells not containing N2-B19-H2b (A2). On dayzero, 45 B10.AKM recipients received 10 Gy total body irradiation (TBI),followed by administration of various concentrations of cultured bonemarrow cells (A2 or H2). K^(b) expression On day 13 several animalsreceiving the lowest doses of cultured bone marrow were sacrificed andindividual spleen colonies were harvested and analyzed by PCR for thepresence of N2-B19-H2b DNA. In addition, spleen cell suspensions wereprepared and analyzed for cell surface expression of K^(b) by flowmicrofluorometry on a fluorescence-activated cell sorter (FACS). FACSanalyses indicated that all animals receiving the H2-treated marrowshowed some Level of K^(b) expression above control staining with thenon-reactive antibody. The results are shown in FIG. 8 which is a FACSprofile of spleen cells from a recipient of transduced bone marrow:A=Anti K^(b) antibody; B=control antibody. Spleen cells from recipientsof non-transduced marrow were also negative. In addition, the PCRanalysis showed every colony examined to contain the transduced DNA.Animals were thereafter followed by FACS and PCR on peripheral bloodlymphocytes (PBL). On day 28 and again on day 40, PCR analyses werepositive. However, FACS analysis for cell-surface expression wasvariable, with PBL from most H2 animals showing only a slight shift ofthe entire peak for staining with anti-K^(b), as compared to PBL from A2animals stained with the same antibody, or as compared to PBL from H2animals stained with the non-reactive HOPC antibody.

Allogeneic grafts On day 40 skin from B10.MBR (K^(b) specific) andB10.BR (control, third party class I disparate) donors was grafted ontoall animals. Graft survivals were scored daily by a blinded observer(i.e., readings were made without knowledge of which graft was fromwhich donor strain) until rejection was complete. The survival times areshown in FIG. 9, and indicate marked specific prolongation of survivalof the B10.MBR skin grafts on the recipients of K^(b)-transduced BMC(FIG. 9B), but not on recipients of control marrow (FIG. 9A). One of theanimals with a long-standing intact B10.MBR skin graft was sacrificed atday 114 and cell suspensions of its lymphoid tissues were examined byFACS and compared to similar suspensions of cells from an animal whichhad rejected its B10.MBR skin graft. A striking difference was noted instaining of thymus cells with an anti-K^(b) mAb. Cell suspensions wereprepared and stained either with the anti-K^(b) mAB 28-8-6 or thecontrol antibody HOPC1. A subpopulation of thymus cells from thetolerant animal showed a marked shift toward increased staining with28-8-6 compared to HOPC1, while there was essentially no change in thestaining pattern of thymocytes from the animal which had lost its graft.FIG. 10 shows FACS analysis on thymocytes from skin graft rejecter(FIGS. 10A, B) and skin graft acceptor (FIGS. 10C, D). Staining withcontrol HOPC1 antibody (FIGS. 10A, C) and with specific anti-K^(b)antibody (FIGS. 10B, D). A similar comparison of staining patterns onbone marrow cells showed the presence of low level K^(b) expression on acell population in the marrow of the tolerant mouse, but not of themouse which had rejected its skin graft. These results indicate that apluripotent stem cell or early progenitor cell population expressedK^(b) in the tolerant mouse but not in the rejecter mouse, and that thisBMC stem cell provided a continuous source of K^(b) antigen in thethymus on cells which are critical for the inactivation of developingthymocytes with K^(b)-reactive TCR. It is of interest to note that K^(b)expression was not detected on splenocytes of the tolerant mouse, andthat, in general, splenocyte expression did not correlate with skingraft tolerance. Since the spleen contains T cells which mature in thethymus, these results suggest that either thymocytes lose expression ofK^(b) as they mature, or that the K^(b)-bearing thymocytes of thisanimal were cells of a non-lymphoid lineage, such as macrophages.

Long-term expression As discussed above, the B10.AKM and B10.MBRcongenic mouse strains are identical except in the MHC class I region. Arecombinant retrovirus containing the class I gene from the B10.MBRstain (H-2K^(b)) linked to a B19 parvovirus promoter (B19-H2K^(b)) and aneomycin resistance (neo^(r)) gene was introduced into B10.AKM(H-2K^(k)) marrow cells. As a control, a recombinant retroviruscontaining only the neo^(r) gene was introduced into B10.AKM marrowcells. The transduced marrow was injected into lethally irradiated AKMrecipients pre-treated with an anti-CD8 monoclonal antibody. Twelveweeks post BMT, quantitative PCR was used to show that the B19-H-2K^(b)proviral sequences were present in 5%-30% of peripheral blood cells inall recipient animals. Reverse transcriptase PCR was used to demonstratethe B19-H-2K^(b) mRNA in RNA isolated from bone marrow and spleen of asubset of recipient animals.

Construction of the K^(b) Retroviral Vector The retroviral vectors usedthe Maloney murine leukemia virus based vector N2, Armentano et al.,1987, J. Virol. 61:1647-1650. The coding regions within this virus weredeleted during its construction, and replaced with the selectable markergene, neomycin phosphotransferase (Neo), which is transcribed from theviral LTR promoter, and provides drug resistance to G418. Thisconventional N2 virus was then further modified by insertion of aparvovirus-derived promoter, B19, Liu et al., 1991, J. Virol. (InPress), downstream from Neo, followed by 1.6 kb of cDNA coding for theclass I antigen H-2K^(b) to form the new recombinant virus N2-B19-H26.FIG. 11 depicts the N2-B19-H26 retroviral vector: P=PstI; X=XhoI;H=HinDIII; E=EcoRI; B=BamHI. This latter cDNA was derived by Waneck etal. during the construction of an H-2^(b) cDNA library for theirpurposes, Waneck et al., 1987, J. Exp. Med. 165:1358-1370.

Viral producer cell lines were developed using the packaging cell linesfor amphotropic (psi-Crip), Danos et al., 1988, Proc. Natl. Acad. Sci85:6460-6464, and ecotropic (psi-2), Sambrook et al., 1989, Molecularcloning: A laboratory manual. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, viral production. These cell lines have been speciallydesigned to produce structural viral proteins for the recombinantdefective virus to be produced. Viral production was achieved bytransfecting psi-Crip with N2-B19-H2b. Both amphotropic and ecotropicproducer cell lines were then co-cultivated allowing multipleintegration events and high expression [i.e. the “ping-pong” techniquesee Bestwick et al., 1988, Proc. Natl. Acad. Sci 85:5404-5408]. In thistechnique, co-cultivation overcomes viral antigen receptor blockage byendogenously secreted proteins since amphotropic and ecotropic virusesrecognize different receptors. Ecotropic psi-2 viral producer cloneswere then selected which produced titers of G418 resistance on 3T3 cellsof greater than 10⁷ cfu/ml.

In order to ensure that K^(b) was being expressed from the recombinantvirus, transduced 3T3 cells were stained with a monoclonal antibodyspecific to this antigen and analyzed by flow microfluorometry. Theseexperiments clearly demonstrated high level expression of virallyderived K^(b).

Animals and husbandry were as follows. The B10.BMR strain, [Sachs etal., 1979, J. Immunol. 123:1965-1969, was provided to the JacksonLaboratory, Bar Harbor, Me. about 6 years ago, and specificpathogen-free stock animals of this strain are now available from thatsource. Upon arrival in animals should be transferred to autoclavedmicroisolator cages containing autoclaved feed and autoclaved acidifieddrinking water. Sterile animal handling procedures which are effectivein maintaining animals free of pathogens so that interpretable survivalstudies can be performed should be used.

Bone marrow transplantation was performed as follows. Techniques forbone marrow transplantation in mice are known to those skilled in theart, see e.g., Sykes et al., 1988, J. Immunol. 140:2903-2911. Briefly,recipient B10.AKM mice aged 12 to 16 weeks are lethally irradiated(1025R, 137Cs source, 110R/min) and reconstituted within 8 hours with2.5×10⁶ bone marrow cells, obtained from the tibiae and femora ofsex-matched donors aged 6-14 weeks. Animals are housed in sterilizedmicroisolator cages, and receive autoclaved food and autoclavedacidified drinking water. For these studies some modifications of thisgeneral technique are required, since the syngeneic bone marrow willhave been transduced with an allogeneic gene, and since the bone marrowwill come from 5FU-treated mice, which should have lower total cellcounts but higher stem cell content than normal mice. The protocol istherefore as follows:

1. Donors will be treated with 5-Fluorouracil, 150 mg/kg i.v. on day −7in order to induce pluripotent stem cells to cycle.

2. Marrow will be harvested from donors on day −5, and T cell depletedwith mAbs and complement.

3. Marrow will than be cultured for 5 days in supernatant from anecotropic packaging cell line (B17H2Kb-18) which produces a high titerof non-infectious retroviral particles containing the K^(b) gene (seebelow). IL-3 and IL-6 will be added to the cultures.

4. On day 0, recipient B10.AKM mice will be lethally irradiated (10.25Gy), and will be reconstituted with 2.5×10⁶ BMC transduced with theK^(b) gene. Control animals will be similarly treated, except that theywill receive marrow exposed to supernatant from a similar ecotropicpackaging line not exposed to a K^(b)-containing vector. The recipientmay also be pre-treated with anti-CD8 monoclonal antibody.

Cellular and serological assays are performed as Follows.

Anti-class I Cell-Mediated Lympholysis (CM) Assay: Spleens are removedfrom BMT recipients and normal mice, red cells are lysed using ACKbuffer, and a single cell suspension is prepared. Cells are filteredthrough 100-mesh nylon, washed, and resuspended at 4×10⁶/ml in completemedium consisting of RPMI 1640 with 10% fetal calf serum, 0.025 mM2-mercapteothanol, 0.01M Hepes buffer, 0.09 mM nonessential amino acids,1 mM sodium pyruvate, 2 mM glutamine, 100 U/ml penicillin and 100 ug/mlstreptomycin. 90 μl of responder cells are added to Costar 96-wellround-bottomed plates along with irradiated (30 Gy) stimulatorsplenocytes. Cultures are set up in two rows of 3 replicates each, andafter 5 days of incubation in 6% CO₂ at 37° C., twofold serial dilutionsare prepared from the second row oftriplicates, so that cytolyticcapacity can be examined at a total of 5 different responder:targetratios. 51Cr-labelled 2-day concanavalin A-induced lymphoblasts are thenadded at 10⁴ blasts per well and incubated for 4 hr at 37° C., 6% CO₂.Plates are harvested using the Titertek supernatant collection system(Skatron, Inc., Sterling, Va.) and ⁵¹Cr release is determined using anautomated gamma counter. Cytolytic capacity is measured directly in theoriginal cell culture plated, so that the measurement is based on thenumber of responders plated, rather than on the number of live cellspresent at the end of the 5-day incubation period. This methodology hasbeen developed and used successfully in this laboratory for severalyears for analysis of spleen cell responses from individual animals[Sykes, M., et al., 1988 J. Immunol. 140:2903-2911]. Percent specificlysis is calculated using the formula:${\%\quad{Specific}\quad{Lysis}} = {\frac{\begin{matrix}{{{Experimental}\quad{release}} -} \\{{Spontaneous}\quad{release} \times 100\quad\%}\end{matrix}}{{{Maximum}\quad{release}} - {{Spontaneous}\quad{release}}} \times 100\quad\%}$Limiting dilution analyses: Responder and stimulator (6×10⁵, 30 Gyirradiated) cells are co-cultured for 7 days in complete mediumcontaining 13% TCGF [lectin-inactivated con A supernatant obtained fromBALB/c con A-Activated splenocytes) in 96-well plates. Wells containing10⁵ (24 wells), 3×10⁴ (24 wells), 10⁴ (30 wells), 3000 (30 wells), 1000(30 wells), 300 (30 wells), and 100 (30 wells) responder cells areprepared. Three thousand ⁵¹Cr-labeled con A blasts are added to eachwell on day 7, and 4 hour ⁵¹Cr release is measured. Wells are consideredpositive if ⁵¹Cr release is 3 standard deviations greater than the mean⁵¹Cr release in 24 wells containing stimulator cells only plus similarnumbers of target cells. The Poisson distribution is used to determinethe frequency of precursor CTL's which recognize each target, andstatistical analysis is performed by the Chi square method of Taswell,Taswell, 1981, J. Immunol. 126:1614.

Flow microfluorometry: One-color and two-color flow cytometry will beperformed, and percentages of cells expressing a particular phenotypewill be determined from 2-color data, as previously described in detailSykes, 1990, J. Immunol. 145:3209-3215. The Lysis II software program(Becton Dickinson) will be used for distinguishing granulocytes fromlymphocytes by gating on the basis of forward angle and 90° lightscatter. Cell sorting will be performed on a Coulter Epics Elite cellsorter. Cell suspensions for flow cytometry: PBL, BMC, thymocyte,splenocyte, and lymph node suspensions will be prepared as previouslydescribed, Sykes, M. et al., 1988, J. Immunol. 140:2903-2911; Sykes, M.1990, J. Immunol. 145:3209-3215; Sharabi, Y. et al., 1990, J. Exp. Med.172:195-202. Whole peripheral white blood cell suspensions (includinggranulocytes) will be prepared by centrifugation of heparinized bloodfor 2 minutes at 14,000 RPM in an Eppendorf centrifuge, followed byaspiration of the buffy coat layer. These cells will be transferred to a15 ml. conical tube and washed. Red blood cells (RBC) contaminating theremaining pellet will be lysed by exposure for 5 seconds to 4.5 ml ofdistilled H₂O followed by rescue with 0.5 ml of 10×PBS.

Cell staining: One-color and two-color staining will be performed as wehave previously described, Sykes, M., 1990, J. Immunol. 145:3209-3215;Sykes et al., 1988, J. Immunol. 141: 2282-2288. Culture supernatant ofrat anti-mouse RcτR mAb 2.4G2, Unkeless, J. C., 1979, J. Exp. Med.150:580-596, will be used for blocking of non-specific staining due toFcτR binding, whenever only direct staining is used. The following mAbsare used: biotinylated murine K^(b)-specific IgG_(2a) mAb 28-8-6, Ozatoet al., 1981, J. Immunol. 126:317-321, and control murine IgG_(2a), mAbHOPC1 (with no known specific binding to murine antigens) are preparedby purification on a protein A-Sepharose column, and are biotinylated bystandard procedures used in our laboratory; rat anti-MAC1 mAb M1/70,Springer et al, 1979, Eur. J. Immunol. 9:301, is used as culturesupernatant, and will be stained by mouse anti-rat IgG-specific mAbMAR18.5; FITC-labeled rat-anti-mouse granulocyte antibody Gr1 ispurchased from Zymed; FITC-labeled rat-anti-mouse Thy1.2 mAb will bepurchased from Becton-Dickinson; FITC-labeled mouse-anti-human CD3 mAbLeu4 (Becton Dickenson) is used as a directly FITC labeled negativecontrol antibody.

Thymic tissue immunofluorescence: The tissue is incubated in L15 mediumfor 24 hours to reduce background staining, and is then cut and embeddedin O.C.T. compound for freezing in Isopentane. Frozen sections areprepared (thickness 4 μm) on a cryostat, dried, fixed in acetone, thenwashed in PBS. The first antibody incubation (with 28-8-6) is performedin the presence of 2% normal mouse serum, in order to saturate Fcreceptors. After 45 minutes, the slides are washed 4 times, andFITC-conjugated secondary reagent (monoclonal rat-anti-mouse IgG2a-FITC,purchased from Pandex) is added. After 45 minutes' incubation with thesecondary reagent, four washes are performed and the tissue is mounted.Sections are examined under a fluorescence microscope by an observer whois unaware of the group of animals from which the tissue was obtained.

Bone Marrow Manipulations and Assays were Performed as Follows:

Transduction of murine bone marrow stem cells: The methodology used fortransduction of bone marrow cells has been described previously,Karlsson et al., 1988, Proc. Natl. Acad. Sci. 85:6062-6066. Bone marrowis harvested from 6-12 week old female B10.AKM donors treated 2 dayspreviously with 150 mg/kg 5-FU. Following T cell depletion (see above),the marrow is divided and 10⁷ cells per 10 cm plate are cultured for 5days in the presence of 8 μg of Polybrene per ml, 10% FCS, 0.6%IL-3-containing supernatant, 0.6% IL-6-containing supernatant, and freshsupernatants from B19H2K^(b) or N2 cells. IL-3- and IL-6-containingsupernatant is 48 hour supernatant of COS 7 cells transfected with themurine rIL-3 gene-containing plasmid pCD-IL-3 or with the murine rIL-6gene-containing plasmid pCD-IL-6, respectively (both plasmids providedby Dr. Frank Lee, DNAX Corp.). IL-3-containing supernatants are titteredby testing proliferation of the IL-3-dependent cell line 32D in thepresence of dilutions of these supernatants, and IL-6 is tittered in asimilar manner using the IL-6-dependent line T1165 as the indicator cellline. We will also test the effect of murine SCF on bone marrowtransduction, as recently described, Zsebo et al., 1990, Cell63:125-201.

The virus-containing supernatants are refreshed on a daily basis byharvesting the non-adherent layer of each plate, pelleting the cells,and resuspending in freshly harvested filtered virus-containingB19H2K^(b) or N2 supernatant with additives. After 5 days, thenon-adherent and adherent BMC are harvested, washed, and resuspended at2.5×10⁶/ml in Medium 199 with Hepes buffer and Gentamycin plus Heparin10 U/Ml. One ml. of this suspension is injected i.v. to irradiated mice.

Murine CFU-GM assay: To test for the bone marrow progenitor cells knownas CFU-GM (colony forming unit-granulocyte/macrophage), bone marrowcells are suspended in plating medium consisting of IMDM mediumcontaining 30% defined fetal bovine serum (FBS) (HyClone, Logan, Utah),10⁻⁴ M β-mercaptoethanol, antibiotics, 5% v/v murine IL-3 culturesupplement (Collaborative Research Inc., Bedford, Mass.) and 0.8%methylcellulose (achieved by adding 36% v/v of a commercially preparedsolution purchased from the Terry Fox Laboratory, Vancouver). 1.1 ml ofthis suspension is then dispensed into 35 mm tissue culture plates (induplicate), and placed in a 37° C. incubator. The resulting CFU-GMderived colonies are enumerated microscopically after 5-7 days.Transduced CFU-GM are selected by including 0.9 μg/ml active G418 in theculture medium. The transduction frequency is then determined by theratio of CFU-GM which form colonies in the presence and in the absenceof the drug.

Molecular methods were as Follows:

Construction of N2-B19-H2b vector: This vector was constructed staringfrom the original retroviral vector N2, Eglitis et al., 1985, Science230:1395-1398, as modified by Shimada to include an additional BamHIsite immediately 3′ of the XhoI site. It includes the K^(b) cDNApreviously cloned in the vector pBG367, as described by G. Waneck,Waneck et al., 1987, J. Exp. Med. 165:1358-1370. This gene has beenplaced under control of the B19 promoter, a highly efficient parvo virusderived promoter, Liu et al., 1991, J. Virol. In Press:] to produce theN2-B19-H2b construct.

Southern blot analysis can be performed on DNA extracted from PBL,thymocyte, BMC, splenocyte or lymph node cell suspensions using standardmethods, Ausubel et al., 1989, Current protocols in molecular biology.John Wiley & Sons, New York, and probing will be performed with thefragment of K^(b) cDNA released from pBG367 by EcoRI. The genomic DNAwill be cut with enzymes capable of distinguishing the transduced K^(b)from other class I genes of the B10.AKM strain. From known sequences itwould appear that EcoRI may be satisfactory for this purpose, since itshould liberate a 1.6 kb band from the transduced K^(b) cDNA, which isdistinct from both the expected endogenous K^(k) and D^(q) class I bandsof B10.AXM, Arnold et al., 1984, Nucl. Acids Res. 12:9473-9485; Lee etal., J. Exp. Med 168:1719-1739. However, to assure that there is noconfusion with bands liberated from other class I and class I-like geneswe will test several enzymes first on DNA from B10.AKM and chooseappropriate restriction enzyme combinations.

PCR analysis of DNA can be performed using primers previously shown tobe effective in our preliminary studies (see FIG. 6): (Seq. ID No. 3)5′ primer: 5′-GGCCCACACTCGCTGAGGTATTTCGTC-3′ (covers 5′ end of α1 exon)(Seq. ID No. 4) 3′ primer: 5′-GCCAGAGATCACCTGAATAGTGTGA-3′ (covers5′ end of α2 exon)

DNA is subjected to 25 cycles of PCR amplification using these specificoligonucleotides and the Cetus GeneAmp kit (Perkin Elmer Cetus, Norwalk,Conn.) according to the manufacturer's directions. In addition,[³²]PdCTP will be included in the PCR reaction in order to visualizeproducts by autoradiography following electrophoresis.

RNA can be isolated from 5×10⁶ to 5×10⁷ cells using the guanidineisothiocyanate and CsCl methods, Chirgwin et al., 1979, Biochem.18:5294-5308, and will be used for northern analyses, RNase protectionanalyses, and for PCR analyses of products formed by reversetranscriptase. For situations in which less then 5×10⁶ cells areavailable, for example following tail bleedings of individual mice, wewill utilize the QuickPrep mRNA Purification Kit (Amgen) as miniaturizedRNA preparation procedure.

Northern analyses can be carried out using standard methods, Ausubel etal., 1989, Current protocols in molecular biology John Wiley & Sons, NewYork, and the same K^(b) cDNA-derived probe. Vector-derived K^(b) mRNAis larger than endogenous class I transcripts (2.5 kb vs. 1.6 kb) due tothe inclusion of vector sequences between the 3′ end of the cDNA and thepoly-adenylation site in the viral 3′ LTR. It should therefore be easyto distinguish the vector-derive K^(b) mRNA from endogenous transcriptsthat might cross-hybridize with a K^(b) cDNA probe. We will also utilizeprobes derived from unique non-K^(b) sequences of the transcript (e.g.,from B19 or N2 derived vector sequences).

RNAse protection analyses-are more sensitive than standard northernblots, yet still quantitative. Procedures based on published methods,Sambrook et al., 1989, Molecular cloning: A laboratory manual. ColdSpring Harbor Laboratory Press, Cold Spring Harbor, will be used toderive riboprobes. Briefly, the Kb cDNA will be cloned into a plasmidvector containing the T3 and T7 RNA polymerase promoter sequences(bluescript or Bluescribe plasmids from Stratagene). Using appropriatepolymerase and ³²P-nucleotides, transcription of the insert will beinitiated and the radioactive K^(b) RNA will be purified. This probewill then be incubated with various RNA preparations followed bytreatment with ribonuclease. Presence of RNA will be assessed byelectrophoresis on a sequencing gel.

PCR following reverse transcriptase treatment of RNA will be used as ahighly sensitive procedure for detecting the K^(b) transcript.Appropriate primers will be designed in order to specifically amplifyretroviral derived transcripts (one primer covering the 5′UT region ofthe construct and second derived from the cDNA sequence). Briefly, RNAwill be prepared by the GuSCN/CsCl method and first strand cDNA will beprepared from 5 ug of total RNA using the SuperScript preamplificationsystem (BRL/Life Technologies, Inc., Gaithersburg, Md.). PCRamplifications will be conducted for 50 cycles, Hansen et al., J.Immunol. 118:1403-1408, using the Cetus GeneAmp kit (Perkin Elmer Cetus,Norwalk, Conn.). Reaction products will be visualized followingelectrophoresis on a 3% NuSieve agarose gel (FMC BioProducts, Rockland,Me.).

Allogeneic MHC Gene Transfer Plus Cyclosporine

It has been shown previously in partially inbred miniature swine thatdifferences in class II MHC loci are of critical importance indetermining the fate of primarily vascularized allografts. Cyclosporinegiven early in the post-transplant period uniformly leads to toleranceof class II matched class I mismatched kidney allografts. However,cyclosporine alone does not produce tolerance across a full-MHC barrier.Consistent with the importance of class II allogeneic bone marrowtransplantation across class II barriers induces tolerance to kidneytransplants matched to the class II of the bone marrow donors, butcompletely disparate to the recipients. In the following experimentspecific transplantation tolerance to complete SLA-disparate kidneytransplant was induced with autologous bone marrow transplantation inwhich the recipient's marrow was genetically modified prior totransplant by transduction with a retroviral expression vector carryingan allogeneic SLA class II gene. The retroviral expression vectorscontained cDNA for either SLA-DRB^(a) or -DRB^(c) and a drug selectionmarker (Neo), and high tittered viral supernatants were prepared usingamphotropic packaging cell lines. Bone marrow from five animals includedin this study was harvested on day 2, and then cultured withvirus-containing supernatant for either an allogeneic (n=4) or syngeneic(n-1) MHC gene for a approximately 48 hours. After lethal irradiation(10 Gy in two fractions 24 hours apart) in days −1 and 0, animals weretransplanted with 0.4 to 1.3×10⁸ cells/kg on day 0. The effectiveness ofthe gene transfer was tested using a colony forming unit assay forgranulocyte/macrophage progenitors (CFU-GM) in the presence of G418 toselect for neomycin resistance. The frequency of G418 resistant CFU-GMvaried significantly between animals (6.5% to 25.9%) immediately aftertransduction and dimnished slowly with time. All animals regained theirresponsiveness to allogeneic stimuli by the third month post-bone marrowtransplantation, as tested by MLR. mAbs specific for DQ and DR moleculesof the class II MHC were used for “blocking” MLR studies, to separatethe effects of recognition of DQ and DR. In assays using cells fromrecipients of bone marrow transplantation transduced with the allogeneicDRB gene, the DR portion of the response to the gene-donor type cellswas strongly diminished, demonstrating the effectiveness of thetransduced gene at inducing DR-specific unresponsiveness. This effectwas observed in all experimental animals, although more pronounced inthe DRB^(d) to cc combination than in DRB^(a) to gg direction. In MRLusing cells from a control animal transduced with a syngeneic gene,blocking of DR or DQ in MLR showed a pattern of reactivity identical tothat observed in naive animals of the same haplotype. Five months afterBMT, animals were challenged with kidney transplants matched for classII of the gene-donor type, and fully mismatched to the originalrecipient haplotype. Cyclosporine 10 to 15 mg/kg/day iv was given for 12days, to tolerize for the class I MHC and minor antigen disparity. Threeanimals rejected their kidney transplants at days 8, 22 and 40. Theaccelerated manner of rejection at day 8 suggested sensitization as anundesirable effect of the expression of the allogeneic gene product. Innone of these recipients could the presence of anti-donor typeantibodies be detected by flow cytometry. One animal became tolerant andexhibited normal creatinine levels at 101 days post-transplant. Theanimal which received the bone marrow transduced with a syngeneic geneunderwent severe rejection with high creatinine levels and vascularchanges in pathology. The recipient of the longest surviving kidneytransplant also received the most efficiently transduced autologous bonemarrow, as judged on the initial frequency of G418r CFU-GM. In this onecase, recombinant cytokines (Pixy 321 (Pixy is a human-GM-CSF/IL3 fusionprotein) 100 Units/ml; mouse stem cell growth factor 20 Units/ml;although these cytokines were used, cytokines from the same species asthe cell being transformed can also be used) were included in theculture medium during transduction with the allogeneic DRB retroviralexpression vector. The cytokines may have led to the transduction ofmultilineage pluripotent hematopoietic stem cells, including theprecursors of dendritic cells which ultimately induced DRB-specifichyporesponsiveness. These experiments demonstrate that somatic class IIMHC DRB gene transfer into bone marrow cells has profound functionalconsequences upon the immune responses of the recipient. In vitro, andmore importantly in vivo, immune function was significantly modulated,with the induction of donor specific prolongation of fully mismatchedkidney transplant survival. The transduction of allogeneic bone marrowstem cells with MHC genes provides a method of inducing tolerance acrossMHC barriers by a mechanism comparable to lymphohematopoietic chimerism.The lethal irradiation used in the experiments described herein can bereplaced with a non-myeloablative conditioning regimen that would permitbone marrow engraftment in a more clinically acceptable fashion.

III. The Induction of Tolerance with Bone Marrow Transplantation

A Short Course of High Dose of Cyclosporine (Administered in Absence ofTreatments. e.g. Treatment with Prednisone which Stimulate CytokineRelease) to Induce Tolerance to Class I and Other Minor DisparitiesCombined with Implantation of Bone Marrow Cells Induce Tolerance toClass II Disparity.

Xenografts: The following procedure was designed to lengthen the time animplanted organ (a xenograft) survives in a xenogeneic host prior torejection. The organ can be any organ, e.g., a liver, e.g., a kidney,e.g., a heart. The main strategies are elimination of natural antibodiesby organ perfusion, transplantation of tolerance-inducing bone marrow,optionally, the implantation of donor stromal tissue, and, optionally,the administration of a short course of a help reducing agent at aboutthe time of introduction of the graft, as described above preparation ofthe recipient for transplantation includes any or all of these steps.Preferably they are carried out in the following sequence.

First, a preparation of horse anti-human thymocyte globulin (ATG) isintravenously injected into the recipient. The antibody preparationeliminates mature T cells and natural killer cells. If not eliminated,mature T cells would promote rejection of both the bone marrowtransplant and, after sensitization, the xenograft itself. Of equalimportance, the ATG preparation also eliminates natural killer (NK)cells. NK cells probably have no effect on the implanted organ, butwould act immediately to reject the newly introduced bone marrow.Anti-human ATG obtained from any mammalian host can also be used, e.g.,ATG produced in pigs, although thus far preparations of pig ATG havebeen of lower titer than horse-derived ATG. ATG is superior to anti-NKmonoclonal Antibodies, as the latter are generally not lytic to all hostNK cells, while the polyclonal mixture in ATG is capable of lysing allhost NK cells. Anti-NK monoclonal antibodies can, however, be used.

The presence of donor antigen in the host thymus during the time whenhost T cells are regenerating post-transplant is critical for tolerizinghost T cells. If donor hematopoietic stem cells are not able to becomeestablished in the host thymus and induce tolerance before host T cellsregenerate repeated doses of anti-recipient T cell antibodies may benecessary throughout the non-myeloablative regimen. Continuous depletionof host T cells may be required for several weeks. Alternatively, e.g.if this approach is not successful, and tolerance (as measured by donorskin graft acceptance, specific cellular hyporesponsiveness in vitro,and humoral tolerance) is not induced in these animals, the approach canbe modified to include host thymectomy. In thymectomized recipients,host T cells do not have an opportunity to differentiate in a hostthymus, but must differentiate in the donor thymus. If this is notpossible, then the animal has to rely on donor T cells developing in thedonor thymus for immunocompetence. Immunocompetence can be measured bythe ability to reject a non-donor type allogeneic donor skin graft, andto survive in a pathogen-containing environment.

It may also be necessary or desirable to splenectomize the recipient inorder to avoid anemia.

Second, the recipient is administered low dose radiation in order tomake room for newly injected bone marrow cells. A sublethal dose ofbetween 100 rads and 400 rads whole body radiation, plus 700 rads oflocal thymic radiation, has been found effective for this purpose.

Third, natural antibodies are adsorbed from the recipient's blood byhemoperfusion of a liver of the donor species. Pre-formed naturalantibodies (nAB) are the primary agents of graft rejection. Naturalantibodies bind to xenogeneic endothelial cells and are primarily of theIgM class. These antibodies are independent of any known previousexposure to antigens of the xenogeneic donor. B cells that produce thesenatural antibodies tend to be T cell-independent, and are normallytolerized to self antigen by exposure to these antigens duringdevelopment. The mechanism by which newly developing B cells aretolerized is unknown. The liver is a more effective adsorber of naturalantibodies than the kidney.

The fourth step in the non-myeloablative procedure is to implant donorstromal tissue, preferably obtained from fetal liver, thymus, and/orfetal spleen, into the recipient, preferably in the kidney capsule. Stemcell engraftment and hematopoiesis across disparate species barriers isenhanced by providing a hematopoietic stromal environment from the donorspecies. The stromal matrix supplies species-specific factors that arerequired for interactions between hematopoietic cells and their stromalenvironment, such as hematopoietic growth factors, adhesion molecules,and their ligands.

As liver is the major site of hematopoiesis in the fetus, fetal livercan also serve as an alternative to bone marrow as a source ofhematopoietic stem cells. The thymus is the major site of T cellmaturation. Each organ includes an organ specific stromal matrix thatcan support differentiation of the respective undifferentiated stemcells implanted into the host. Although adult thymus may be used, fetaltissue obtained sufficiently early in gestation is preferred because itis free from mature T lymphocytes which can cause GVHD. Fetal tissuesalso tend to survive better than adult tissues when transplanted. As anadded precaution against GVHD, thymic stromal tissue can be irradiatedprior to transplantation, e.g., irradiated at 1000 rads. As analternative or an adjunct to implantation, fetal liver cells can beadministered in fluid suspension.

Fifth, bone marrow cells (BMC), or another source of hematopoietic stemcells, e.g., a fetal liver suspension, of the donor are injected intothe recipient. Donor BMC home to appropriate sites of the recipient andgrow contiguously with remaining host cells and proliferate, forming achimeric lymphohematopoietic population. By this process, newly formingB cells (and the antibodies they produce) are exposed to donor antigens,so that the transplant will be recognized as self. Tolerance to thedonor is also observed at the T cell level in animals in whichhematopoietic stem cell, e.g., BMC, engraftment has been achieved. Whenan organ graft is placed in such a recipient several months after bonemarrow chimerism has been induced, natural antibody against the donorwill have disappeared, and the graft should be accepted by both thehumoral and the cellular arms of the immune system. This approach hasthe added advantage of permitting organ transplantation to be performedsufficiently long following transplant of hematopoietic cells, e.g.,BMT, e.g., a fetal liver suspension, that normal health andimmunocompetence will have been restored at the time of organtransplantation. The use of xenogeneic donors allows the possibility ofusing bone marrow cells and organs from the same animal, or fromgenetically matched animals.

Finally, a short course of a help reducing agent, e.g., a short courseof high dose CsA is administered to the recipient. As is describedabove, the course is begun at about the time of implantation, or alittle before, and is continued for a time about equal to the time ittakes for a mature T cell to be stimulated and initiate rejection. Whileany of these procedures may aid the survival of an implanted organ, bestresults are achieved when all steps are used in combination. Methods ofthe invention can be used to confer tolerance to allogeneic grafts,e.g., wherein both the graft donor and the recipient are humans, and toxenogeneic grafts, e.g., wherein the graft donor is a nonhuman animal,e.g., a swine, e.g., a miniature swine, and the graft recipient is aprimate, e.g., a human.

While any of these procedures may aid the survival of an implantedorgan, best results are achieved when all steps are used in combination.Methods of the invention can be used to confer tolerance to allogeneicgrafts, e.g., wherein both the graft donor and the recipient are humans,and to xenogeneic grafts, e.g., wherein the graft donor is a nonhumananimal, e.g., a swine, e.g., a miniature swine, and the graft recipientis a primate, e.g., a human.

In the case of xenogeneic grafts, the donor of the implant and theindividual that supplies either the tolerance-inducing hematopoieticcells or the liver to be perfused should be the same individual orshould be as closely related as possible. For example, it is preferableto derive implant tissue from a colony of donors that is highly inbred.

Detailed Protocol

In the following protocol for preparing a cynomolgus monkey for receiptof a kidney from a miniature swine donor, zero time is defined as themoment that the arterial and venous cannulas of the recipient areconnected to the liver to be perfused.

On day −1 a commercial preparation (Upjohn) of horse anti-humananti-thymocyte globulin (ATG) is injected into the recipient. ATGeliminates mature T cells and natural killer cells that would otherwisecause rejection of the bone marrow cells used to induce tolerance. Therecipient is anesthetized, an IV catheter is inserted into therecipient, and 6 ml of heparinized whole blood are removed beforeinfection. The ATG preparation is then injected (50 mg/kg)intravenously. Six ml samples of heparinized whole blood are drawn fortesting at time points of 30 min., 24 hours and 48 hours. Blood samplesare analyzed for the effect of antibody treatment on natural killer cellactivity (testing on K562 targets) and by FACS analysis for lymphocytesubpopulations, including CD4, CD8, CD3, CDllb, and CD16. Preliminarydata from both assays indicate that both groups of cells are eliminatedby the administration of ATG. If mature T cells and NK cells are noteliminated, ATG can be re-administered at later times in the procedure,both before and after organ transplantation.

Sublethal irradiation is administered to the recipient between days −1and −8. Irradiation is necessary to eliminate enough of the recipient'sendogenous BMC to stimulate hematopoiesis of the newly introducedforeign BMC. Sublethal total body irradiation is sufficient to permitengraftment with minimal toxic effects to the recipient. Whole bodyradiation (150 Rads) was administered to cynomolgus monkey recipientsfrom a bilateral (TRBC) cobalt teletherapy unit at 10 Rads/min. Localirradiation of the thymus (700 Rads) was also employed in order tofacilitate engraftment.

Natural antibodies are a primary cause of organ rejection. To removenatural antibodies from the recipient's circulation prior totransplantation, on day 0 an operative adsorption of natural antibodies(nAB) is performed, using a miniature swine liver, as follows. At −90minutes the swine donor is anesthetized, And the liver prepared forremoval by standard operative procedures. At −60 minutes the recipientmonkey is anesthetized. A peripheral IV catheter is inserted, and a 6 mlsample of whole blood is drawn. Through mid-line incision, the abdominalaorta and the vena cava are isolated. Silastic cannulas containing sideports for blood sampling are inserted into the blood vessels.

At −30 minutes the liver is perfused in situ until it turns pale, andthen removed from the swine donor and placed into cold Ringers Lactate.The liver is kept cold until just prior to reperfusion in the monkey. Aliver biopsy is taken. At −10 minutes the liver is perfused with warmalbumin solution until the liver is warm (37 degrees).

At 0 time the arterial and venous cannulas of the recipient areconnected to the portal vein and vena cava of the donor liver andperfusion is begun. Liver biopsies are taken at 30 minutes and 60minutes, respectively. Samples of recipient blood are also drawn forserum at 30 minutes and 60 minutes respectively. At 60 minutes the liveris disconnected from the cannulas and the recipient's large bloodvessels are repaired. The liver, having served its function of adsorbingharmful natural antibodies from the recipient monkey, is discarded.Additional blood samples for serum are drawn from the recipient at 2,24, and 48 hours. When this procedure was performed on two sequentialperfusions of swine livers, the second liver showed no evidence of mildischemic changes during perfusion. At the end of a 30 minute perfusionthe second liver looked grossly normal and appeared to be functioning,as evidenced by darkening of the venous outflow blood compared to thearterial inflow blood in the two adjacent cannulas. Tissue sections fromthe livers were normal, but immunofluorescent stains showed IgM onendothelial cells. Serum samples showed a decrease in naturalantibodies.

To promote long-term survival of the implanted organ through T-cell andB-cell mediated tolerance, donor bone marrow cells are administered tothe recipient to form chimeric bone marrow. The presence of donorantigens in the bone marrow allows newly developing B cells, and newlysensitized T cells, to recognize antigens of the donor as self, andthereby induces tolerance for the implanted organ from the donor. Tostabilize the donor BMC, donor stromal tissue, in the form of tissueslices of fetal liver, thymus, and/or fetal spleen are transplantedunder the kidney capsule of the recipient. Stromal tissue is preferablyimplanted simultaneously with, or prior to, administration ofhematopoietic stem cells, e.g., BMC, or a fetal liver cell suspension.

To follow chimerism, two color flow cytometry can be used. This assayuses monoclonal antibodies to distinguish between donor class I majorhistocompatibility antigens and leukocyte common antigens versusrecipient class I major histocompatibility antigens. BMC can in turn beinjected either simultaneously with, or preceding, organ transplant.Bone marrow is harvested and injected intravenously (7.5×10⁸/kg) aspreviously described (Pennington et al., 1988, Transplantation45:21-26). Should natural antibodies be found to recur before toleranceis induced, and should these antibodies cause damage to the graft, theprotocol can be modified to permit sufficient time following BMT forhumoral tolerance to be established prior to organ grafting.

The approaches described above are designed to synergistically preventthe problem of transplant rejection. When a kidney is implanted into acynomolgus monkey following liver adsorption of natural antibodies,without use of bone marrow transplantation to induce tolerance, renalfunctions continued for 1-2 days before rejection of the kidney. Whenfour steps of the procedure were performed (adsorption of naturalantibodies by liver perfusion, administration of ATG, sublethalirradiation and bone marrow infusion, followed by implant of a porcinekidney into primate recipient), the kidney survived 7 days beforerejection. Despite rejection of the transplanted organ, the recipientremained healthy.

When swine fetal liver and thymic stromal tissue were implanted underthe kidney capsule of two sublethally irradiated SCID mice, 25-50% ofperipheral blood leukocytes were of donor lineage two weekspost-transplantation. A significant degree of chimerism was not detectedin a third animal receiving fetal liver without thymus.

The methods of the invention may be employed in combination, asdescribed, or in part.

The method of introducing bone marrow cells may be altered, particularlyby (1) increasing the time interval between injecting hematopoietic stemcells and implanting the graft; (2) increasing or decreasing the amountof hematopoietic stem cells injected; (3) varying the number ofhematopoietic stem cell injections; (4) varying the method of deliveryof hematopoietic stem cells; (5) varying the tissue source ofhematopoietic stem cells, e.g., a fetal liver cell suspension may beused; or (6) varying the donor source of hematopoietic stem, cells.Although hematopoietic stem cells derived from the graft donor arepreferable, hematopoietic stem cells may be obtained from otherindividuals or species, or from genetically-engineered inbred donorstrains, or from in vitro cell culture.

Methods of preparing the recipient for transplant of hematopoietic stemcells may be varied. For instance, recipient may undergo a splenectomyor a thymectomy. The latter would preferably be administered prior tothe non-myeloablative regimen, e.g., at day −14.

Hemoperfusion of natural antibodies may: (1) make use of other vascularorgans, e.g., liver, kidney, intestines; (2) make use of multiplesequential organs; (3) vary the length of time each organ is perfused;(4) vary the donor of the perfused organ. Irradiation of the recipientmay make use of: (1) varying the absorbed dose of whole body radiationbelow the sublethal range; (2) targeting different body parts (e.g.,thymus, spleen); (3) varying the rate of irradiation (e.g., 10 rads/min,15 rads/min); or (4) varying the time interval between irradiation andtransplant of hematopoietic stem cells; any time interval between 1 and14 days can be used, and certain advantages may flow from use of a timeinterval of 4-7 days. Antibodies introduced prior to hematopoietic celltransplant may be varied by: (1) using monoclonal antibodies to T cellsubsets or NK cells (e.g., anti-NKH1_(A), as described by U.S. Pat. No.4,772,552 to Hercend, et al., hereby incorporated by reference); (2)preparing anti-human ATG in other mammalian hosts (e.g., monkey, pig,rabbit, dog); or (3) using anti-monkey ATG prepared in any of the abovementioned hosts.

The methods of the invention may be employed with other mammalianrecipients (e.g., rhesus monkeys) and may use other mammalian donors(e.g., primates, sheep, or dogs). As an alternative or adjunct tohemoperfusion, host antibodies can be depleted by administration of anexcess of hematopoietic cells.

Stromal tissue introduced prior to hematopoietic cell transplant, e.g.,BMT, may be varied by: (1) administering the fetal liver and thymustissue as a fluid cell suspension; (2) administering fetal liver orthymus stromal tissue but not both; (3) placing a stromal implant intoother encapsulated, well-vascularized sites, or (4) using adult thymusor fetal spleen as a source of stromal tissue.

Tolerance to fully MHC mismatched renal allografts in chimeric swine

Overwhelming importance of major histocompatibility complex (MHC) classII matching for achieving tolerance of kidney transplants (KTx) inminiature swine has been demonstrated previously. When class II antigensare matched, long-term specific tolerance across MHC class I and minorantigens (MA) barrier, can uniformly be induced by a short course ofcyclosporine. However, cyclosporine does not produce this effect acrossa full MHC barrier. Bone marrow transplantation (BMT) acrosssingle-haplotype class II MHC+MA barriers creates fully chimericanimals, as confirmed by FCM. These chimeras recover normal cellularimmune function 2-3 months after BMT, as tested by MLR and CML. Foursuch chimeric animals (see Table V, numbers 1-4) received kidneytransplants from donors class II matched to BMT donors and fullymismatched to the recipients. A 12-day course of cyclosporine (10mg/kg/day) was the only immunosuppression following kidneytransplantation. All 4 pigs have maintained normal creatinine (Cr)values (<2 mg %) for longer than 300 days, and one recipient is aliveover 3 years with good kidney function (Cr<2 mg %) and graft histologyshowing minimal borderline rejection. These results demonstrate thatinduction of tolerance to class II antigens by BMT allows a short courseof cyclosporine to induce specific tolerance (as tested by skin grafts)to fully allogeneic kidney transplants. Subsequently, we have examinedthe specificity of this phenomenon by determining if single-haplotypeclass II+MA mismatched BMT will facilitate cyclosporine inducedlong-term acceptance of kidney transplants completely mismatched to boththe recipient and BMT donor (Table V, numbers 5-10). A 12-day course ofcyclosporine allowed long-term survival of such kidney transplants inchimeric recipients. Animal #5 was still alive and clinically well, withnormal Cr levels; histology however reveals borderline rejection. Animal#6 was sacrificed 18 months after kidney transplant, with deterioratingkidney function (Cr>11 mg %). Animal #7 was sacrificed at 6 months afterkidney transplant due to sepsis, kidney transplants showed moderatetubulointestinal infiltrate without signs of vascular injury. Bothlong-term survivors (pigs #3 & 5) were recently tested for anti-donorreactivity. CML and MLR revealed specific unresponsiveness to the kidneytransplant donor type cells. Pigs #8-10 received kidney transplant fromoutbred Yorkshire donors. These animals developed irreversible renalfailure, starting shortly after cessation of the cyclosporine therapy.TABLE V # Recipient BMT Donor KTx Donor Outcome (funct./pathol.) 1 aa(I^(aa)II^(aa)) aj (I^(aa)II^(ac)) cc (I^(cc)I^(cc)) sac 1 y(good/normal) 2 ac (I^(ac)II^(ac)) ag (I^(ac)II^(ad)) dd (I^(dd)II^(dd))died >2.5 y (good/ chronic rej) 3 ac (I^(ac)II^(ac)) ag (I^(ac)II^(ad))dd (I^(dd)II^(dd)) alive >3 y (good/border rej) 4 ac (I^(ac)II^(ac)) ag(I^(ac)II^(ad)) dd (I^(dd)II^(dd)) sac 1 y (good/normal) 5 aa(I^(aa)II^(aa)) ah (I^(aa)II^(ad)) cc (I^(cc)II^(cc)) alive >2.5 y(good/border rej) 6 aa (I^(aa)II^(aa)) ah (I^(aa)II^(ad)) cc(I^(cc)II^(cc)) sac >1.5 y (poor/chronic rej) 7 aa (I^(aa)II^(aa)) aj(I^(aa)II^(ac)) dd (I^(dd)II^(dd)) sac 0.5 y (good/infiltrate) 8 aa(I^(aa)II^(aa)) aj (I^(aa)II^(ac)) YORK (I^(?)II^(?)) sac 30 d(poor/acute rej) 9 ac (I^(ac)II^(ac)) ch (I^(ac)II^(ad)) YORK(I^(?)II^(?)) sac 70 d (poor/acute rej) 10 ac (I^(ac)II^(ac)) ch(I^(ac)II^(ad)) YORK (I^(?)II^(?)) sac 38 d (poor/acute rej)sac = sacrificed;rej = rejection

Thus, a short postoperative course of cyclosporine in MHC class IImismatched BMT recipients allows tolerance to be induced to kidneytransplants that are class II matched to the BMT donor. Long-termunresponsiveness to kidney transplants that are fully mismatched to boththe recipient and BMT donor can be achieved in some cases, apparentlydependent on the degree of disparity at multiple loci (compare with thedifference between inbred and outbred donors).

A Short Course of Cyclosporine to Suppress T Cell Function in PrimateAllogeneic Kidney Transplantation.

The following experiment shows that mixed chimerism, obtained during anon-myeloablative protocol to achieve engraftment, is capable ofproducing multilineage lymphohematopoietic chimerism and long-termtolerance to renal allografts between fully MHC mismatched cynomolgusmonkeys. Complete ablation of host lymphohematopoietic elements isneither necessary nor desirable when bone marrow transplantation isutilized as a tolerance-inducing regimen. Instead, it is advantageous toachieve a state of mixed chimerism, in which the presence of certaindonor-derived elements induce specific tolerance, while host-typeantigen presenting cells maintain normal immunocompetence.

It has been demonstrated in murine studies that removal of mature host Tcells is important in order to achieve mixed chimerism. In initialstudies using fully MHC mismatched cynomolgus monkeys, a variety ofmonoclonal antibodies were tested to mature T cell subsets (anti-CD4 andanti-CD8) as well as several sources of anti-thymocyte globulin (ATG) asT cell depleting reagents. Although these antibody treatments led tomarked depletion of T cells in the peripheral blood, biopsies of lymphnodes demonstrated that residual T cells remained, often coated withantibody. In order to further suppress T cell function, a one-monthcourse of treatment with an i.m. preparation of cyclosporine(CyA) in oilwas added to the preparative regimen. This treatment led to therapeuticlevels of cyclosporine during drug administration and to tapering levelsover a period of 3 weeks after the drug was discontinued. The basicprotocol for nonlethal preparative regimen was as follows: Cynomolgusmonkeys weighing 6 to 10 kg. (Charles River Primates, Wilmington, Mass.)were treated with 300 Rads of WBI either as a single dose (#M393) on day−6 or as two fractions of 150 Rads each on days −6 and −5 (#M3093 and#M3293). 700 Rads of thymic irradiation was administered on day −1.Horse anti-human thymocyte globulin (ATG) (Upjohn) was administered at50 mg/kg i.m. on days −2, −1 and 0. Orthotopic kidney transplantationwas performed on day 0 through a midline incision using end to sideanastamoses of the donor renal artery and renal vein into the recipientaorta and vena cava, respectively, and using a ureteroureteralanastomosis for urinary drainage. Bone marrow was harvested from twodonor ribs, prepared as a single cell suspension, and infused i.v. intothe recipient at the end of the renal transplant. Treatment withcyclosporine (Sandimmune®, 15 mg/kg/day, suspended in olive oil) i.m.was begun on day 0 and continued for 27 days.

Monkey #393 became pancytopenic on day 8, and required three bloodtransfusions with blood group matched, irradiated whole blood over thenext two weeks. However, peripheral blood components recovered graduallythereafter, and were normal by day 30. Renal function has remainednormal for over 250 days, and a biopsy on day 215 showed a normalkidney.

Sequential flow cytometric (FCM) analyses were performed on this animalutilizing a monoclonal anti-class I antibody previously determined todistinguish donor from host, and analyzing lymphoid, monocytic andneutrophil populations as determined by scatter profiles. Clear evidencefor chimerism in all three subpopulations was detected first on day 10,and persisted at similarly high levels until cyclosporine treatment wasdiscontinued on day 27. Thereafter, the levels of chimerism detected ineach subpopulation decreased, but chimerism was still detectable by FCMamong lymphocytes (1.5%) and monocytes (29%) as late as day 203, thelast day tested. In addition, a bone marrow aspirate on day 203 showed11.2% donor cells by FCM.

Mixed lymphocyte reactions performed pre-transplant and on day 159post-transplant revealed a specific loss of anti-donor reactivity (TableVI). TABLE VI 3^(rd) Time Medium Autologous Donor Party #1 3^(rd) Party#2 Pre- 888 2434 5946 5571 6986 Transplant (CPM) Pre- — 1.0 3.3 3.0 3.9Transplant (Stim. Index) Day 159 703 3410 2324 11298 9127 (CPM) Day 159— 1.0 0.6 4.2 3.1 (Stim. Index)

This result, combined with normal renal function and normal kidneyhistology without any additional exogenous immunosuppression since day27, lead us to conclude that specific transplantation tolerance has beeninduced in this animal through the establishment of mixed chimerism. Twoadditional animals were treated by the same protocol, but with anintravenous preparation of cyclosporine which led to an abrupt fall ofcyclosporine levels in the blood after discontinuation rather thangradual tapering of levels over a three-week period. One of theseanimals died of sepsis on day 12 during the period of aplasia, and theother lost evidence for chimerism after discontinuation of cyclosporineand although still alive on day 100, has shown a course consistent withchronic rejection both by clinical and pathological criteria.

In order to reduce the toxicity of the preparative regimen, we havesubsequently modified the irradiation protocol. In one animal (#3893)the WBI was decreased to 1.5 Gy. This animal failed to develop mixedchimerism and rejected the kidney transplant (Creatinine=12.1 on day47). In two additional animals (#3093 and #3292) the WBI was maintainedat 3.0 Gy, but was fractionated to 1.5 Gy on two successive days (−6 and−5) rather than administered as a single dose. Both of these animalsdeveloped mixed multilineage chimerism, first detectable on day 11 andday 20 respectively. They showed much less toxicity from the preparativeregimen than did the animals receiving unfractionated irradiation, andboth remain chimeric with normal renal function at the time of thiswriting (day 40 and day 25, respectively).

Pig to Monkey Kidney Xenotransplantation by a Mixed Chimerism Approach

The following experiment shows the induction of tolerance in monkeys topig organs by means of a xenogeneic lymphohematopoietic chimerismapproach which has previously been shown effective in concordant rodentsystems. To date 16 Cynomolgus monkeys have received pig kidneytransplants along with xenogeneic bone marrow from the same donor. Thepreparative regimen for these xenografts included: 1) conditioning withnon-myeloablative whole body irradiation (WBI) and thymic irradiation;2) removal of preformed mAbs by perfusion of monkey blood through a pigliver; 3) splenectomy; 4) T cell depletion with ATG and/or mAbs; and 5)postoperative immunosuppression with cyclosporine and in some animalsanti-IgM mAbs. Ten animals have survived more than 4 days, with thelongest surviving 13 days, with normal renal function to day 11. In thisanimal pig cells were detected in the peripheral blood only at day 10post-transplant, suggesting transient xenogeneic chimerism. Two monkeysreceived only splenectomy and pig liver perfusion prior to the kidneyxenograft. In one of these animals, in which no furtherimmunosuppression was administered post-transplant, the kidneyfunctioned for 3 days, then rapidly lost function, with completerejection by day 5. Analysis of this monkey's sera by flow cytometryindicated return of high titers of IgM, which correlated with rejection.In the second animal cyclosporine 15 mg/kg/day iv and 15 deoxyspergualin(DSG) 6 mg/kg/day iv were administered post-transplant. The kidneyfunctioned until day 7, then failed and was removed on day 8. Pathologicexamination showed a focal inflammatory infiltrate in addition to patchyinterstitial hemorrhage. The infiltrate contained approximately 20% Tcells, as determined by staining with mAbs to CD3, CD4 and CD8. IgMnatural antibodies were effectively removed during liver perfusion inthis animal, and strikingly, they did not appear in the serumthereafter, IgG levels started to rise on day 7, correlating with thebeginning of renal dysfunction. These results show 1) that naturalantibody (IgM) responses can be effectively eliminated by components ofthe preparative regimen involving pig liver adsorption andpost-operative suppression with DSG; and 2) that T cell suppressivecomponents of the preparative regimen (i.e., irradiation, cyclosporineand ATG) are required to prevent cellular and secondary (IgG) responsesin these experiments.

Other Embodiments

Stromal tissue introduced prior to hematopoietic cell transplant, e.g.,BMT, may be varied by: (1) administering the fetal liver and thymustissue as a fluid cell suspension; (2) administering fetal liver orthymus stromal tissue but not both; (3) placing a stromal implant intoother encapsulated, well-vascularized sites, or (4) using adult thymusor fetal spleen as a source of stromal tissue.

The methods described herein for inducing tolerance to, or promoting theacceptance of, an allogeneic antigen or allogeneic graft can be usedwhere, as between the donor and recipient, there is any degree ofmismatch at MHC loci or other loci which influence graft rejection.Preferably, there is a mismatch at least one MHC locus or at least oneother locus that mediates recognition and rejection, e.g., a minorantigen locus. With respect to class I and class II MHC loci, the donorand recipient can be: matched at class I and mismatched at class II;mismatched at class I and matched at class II; mismatched at class I andmismatched at class II; matched at class I, matched at class II. In anyof these combinations other loci which control recognition andrejection, e.g., minor antigen loci, can be matched or mismatched. Asstated above, it is preferable that there is mismatch at least onelocus. Mismatched at MHC class I means mismatched for one or more MHCclass I loci, e.g., in the case of humans, mismatched at one or more ofHLA-A, HLA-B, or HLA-C, or in the case of swine, mismatch at one or moreSLA class I loci, e.g., the swine A or B loci. Mismatched at MHC classII means mismatched at one or more MHC class II loci, e.g., in the caseof humans, mismatched at one or more of a DP α, a DPβ, a DQ α, a DQ β, aDR α, or a DR β, or in the case of swine, mismatch at one or SLA classII loci, e.g., mismatch at DQ α or β, or DR α or β.

The methods described herein for inducing tolerance to an allogeneicantigen or allogeneic graft can be used where, as between the donor andrecipient, there is any degree of reactivity in a mixed lymphocyteassay, e.g., wherein there is no, low, intermediate, or high mixedlymphocyte reactivity between the donor and the recipient. In preferredembodiments mixed lymphocyte reactivity is used to define mismatch forclass II, and the invention includes methods for performing allogeneicgrafts between individuals with any degree of mismatch at class II asdefined by a mixed lymphocyte assay. Serological tests can be used todetermine mismatch at class I or II loci and the invention includesmethods for performing allogeneic grafts between individuals with anydegree of mismatch at class I and or II as measured with serologicalmethods. In a preferred embodiment, the invention features methods forperforming allogeneic grafts between individuals which, as determined byserological and or mixed lymphocyte reactivity assay, are mismatched atboth class I and class II.

The methods of the invention are particularly useful for replacing atissue or organ afflicted with a neoplastic disorder, particularly adisorder which is resistant to normal modes of therapy, e.g.,chemotherapy or radiation therapy. Methods of the invention can be usedfor inducing tolerance to a graft, e.g., an allograft, e.g., anallograft from a donor which is mismatched at one or more class I lociat one or more class II loci or at one or more loci at each of class Iand class II. In preferred embodiments: the graft includes tissue fromthe digestive tract or gut, e.g., tissue from the stomach, or boweltissue, e.g., small intestine, large intestine, or colon; the graftreplaces a portion of the recipient's digestive system e.g., all or partof any of the digestive tract or gut, e.g., the stomach, bowel, e.g.,small intestine, large intestine, or colon.

Tolerance, as used herein, refers not only to complete immunologictolerance to an antigen, but to partial immunologic tolerance, i.e., adegree of tolerance to an antigen which is greater than what would beseen if a method of the invention were not employed.

As is discussed herein, it is often desirable to expose a graftrecipient to irradiation in order to promote the development of mixedchimerism. The inventor has discovered that it is possible to inducemixed chimerism with less radiation toxicity by fractionating theradiation dose, i.e., by delivering the radiation in two or moreexposures or sessions. Accordingly, in any method of the inventioncalling for the irradiation of a recipient, e.g., a primate, e.g., ahuman, recipient, of a xenograft or allograft, the radiation can eitherbe delivered in a single exposure, or more preferably, can befractionated into two or more exposures or sessions. The sum of thefractionated dosages is preferably equal, e.g., in rads or Gy, to theradiation dosage which can result in mixed chimerism when given in asingle exposure. The fractions are preferably approximately equal indosage. For example, a single dose of 700 rads can be replaced with,e.g., two fractions of 350 rads, or seven fractions of 100 rads.Hyperfractionation of the radiation dose can also be used in methods ofthe invention. The fractions can be delivered on the same day, or can beseparated by intervals of one, two, three, four, five, or more days.Whole body irradiation, thymic irradiation, or both, can befractionated.

The inventor has also discovered that much or all of the preparativeregimen can be delivered or administered to a recipient, e.g., anallograft or xenograft recipient, within a few days, preferably within72, 48, or 24 hours, of transplantation of tolerizing stem cells and/orthe graft. This is particularly useful in the case of humans receivinggrafts from cadavers. Accordingly, in any of the methods of theinvention calling for the administration of treatments prior to thetransplant of stem cells and/or a graft, e.g., treatments to inactivateor deplete host antibodies, treatments to inactivate host T cells or NKcells, or irradiation, the treatment(s) can be administered, within afew days, preferably within 72, 48, or 24 hours, of transplantation ofthe stem cells and/or the graft. In particular, primate, e.g., human,recipients of allografts can be given any or all of treatments toinactivate or deplete host antibodies, treatments to inactivate host Tcells or NK cells, or irradiation, within a few days, preferably within72, 48, or 24 hours, of transplantation of stem cells and/or the graft.For example, treatment to deplete recipient T cells and/or NK cells,e.g., administration of ATG, can be given on day −2, −1, and 0, and WBI,thymic irradiation, and stem cell, e.g., bone marrow stem cells,administered on day 0. (The graft, e.g., a renal allograft, istransplanted on day 0).

Methods of the invention can include recipient splenectomy.

As is discussed herein, hemoperfusion, e.g., hemoperfusion with a donororgan, can be used to deplete the host of natural antibodies. Othermethods for depleting or otherwise inactivating natural antibodies canbe used with any of the methods described herein. For example, drugswhich deplete or inactivate natural antibodies, e.g., deoxyspergualin(DSG) (Bristol), or anti-IgM antibodies, can be administered to therecipient of an allograft or a xenograft. One or more of, DSG (orsimilar drugs), anti-IgM antibodies, and hemoperfusion, can be used todeplete or otherwise inactivate recipient natural antibodies in-methodsof the invention. DSG at a concentration of 6 mg/kg/day, i.v., has beenfound useful in suppressing natural antibody function in pig tocynomolgus kidney transplants.

Some of the methods described herein use lethal irradiation to createhematopoietic space, and thereby prepare a recipient for theadministration of allogeneic, xenogeneic, syngeneic, or geneticallyengineered autologous, stem cells. In any of the methods describedherein, particularly primate or clinical methods, it is preferable tocreate hematopoietic space for the administration of such cells bynon-lethal means, e.g., by administering sub-lethal doses ofirradiation, bone marrow depleting drugs, or antibodies. The use ofsublethal levels of bone marrow depletion allows the generation of mixedchimerism in the recipient. Mixed chimerism is generally preferable tototal or lethal ablation of the recipient bone marrow followed bycomplete reconstitution of the recipient with administered stem cells.

Alternative methods for the inactivation of thymic T cells are alsoincluded in embodiments of the invention. Some of the methods describedherein include the administration of thymic irradiation to inactivatehost thymic-T cells or to otherwise diminish the host's thymic-T cellmediated responses to donor antigens. It has been discovered that thethymic irradiation called for in allogeneic or xenogeneic methods of theinvention can be supplemented with, or replaced by, other treatmentswhich diminish (e.g., by depleting thymic-T cells and/or down modulatingone or more of the T cell receptor (TCR), CD4 co-receptor, or CD8co-receptor) the host's thymic-T cell mediated response. For example,thymic irradiation can be supplemented with, or replaced by, anti-T cellantibodies (e.g., anti-CD4 and/or anti-CD8 monoclonal antibodies)administered a sufficient number of times, in sufficient dosage, for asufficient period of time, to diminish the host's thymic-T cell mediatedresponse.

For best results, anti-T cell antibodies should be administeredrepeatedly. E.g., anti-T cell antibodies can be administered one, two,three, or more times prior to donor bone marrow transplantation.Typically, a pre-bone marrow transplantation dose of antibodies will begiven to the patient about 5 days prior to bone marrow transplantation.Additional, earlier doses 6, 7, or 8 days prior to bone marrowtransplantation can also be given. It may be desirable to administer afirst treatment then to repeat pre-bone marrow administrations every 1-5days until the patient shows excess antibodies in the serum and about99% depletion of peripheral T cells and then to perform the bone marrowtransplantation. Anti-T cell antibodies can also be administered one,two, three, or more times after donor bone marrow transplantation.Typically, a post-bone marrow transplant treatment will be given about2-14 days after bone marrow transplantation. The post bone marrowadministration can be repeated as many times as needed. If more than oneadministration is given the administrations can be spaced about 1 weekapart. Additional doses can be given if the patient appears to undergoearly or unwanted T cell recovery. Preferably, anti-T cell antibodiesare administered at least once (and preferably two, three, or moretimes) prior to donor bone marrow transplantation and at least once (andpreferably two, three, or more times) after donor bone marrowtransplantation.

The following experiments show that additional T cell-depletingantibodies can replace thymic irradiation in a non-myeloablativeconditioning regimen and allow allogeneic bone marrow engraftment anddonor-specific tolerance induction.

A low toxicity, non-myeloablative conditioning regimen that allowsallogeneic bone marrow engraftment and donor-specific toleranceinduction in mice has been previously described. A regimen whichincludes pre-treatment with depleting doses of anti-CD4 and anti-CD8monoclonal antibodies on day −5, administration of 3 Gy whole bodyirradiation and 7 Gy of thymic irradiation on day 0 followed byadministration of fully MHC-mismatched donor bone marrow cells, allowsthe induction of permanent mixed chimerism and skin graft tolerance. Thethymic irradiation step in this protocol was replaced with additionalanti-CD4 and anti-CD8 monoclonal antibody treatment. Multilineagechimerism was compared in B10 (H-2^(b)) mice receiving allogeneic(B10.A, H-2^(a)) bone marrow transplantation on day 0 following 3 Gywhole body irradiation with or without thymic irradiation, and treatmentwith monoclonal antibodies by a variety of schedules pre and post-bonemarrow transplantation. Most (50 of 52) animals that either receivedthymic irradiation or that received at least two pre-bone marrowtransplantation monoclonal antibody treatments demonstrated long-termmultilineage peripheral blood mixed allogeneic chimerism (asdemonstrated by flow cytometric analysis). In contrast, only 1 of 8animals receiving only one pre-bone marrow transplantation monoclonalantibody treatment without thymic irradiation developed lasting (morethan 20 weeks) mixed chimerism. AU chimeric animals accepted donor skingrafts for more than 100 days and rejected third party BALB/c graftswithin 14 days. Therefore, mixed chimerism and donor-specific skin graftacceptance could be induced without the use of thymic irradiation if atleast 2 pre-bone marrow transplantation monoclonal antibody treatmentswere given. (The monoclonal antibody treatments were spaced about 5 daysapart with the final treatment 1 day prior to bone marrowtransplantation.) However, levels of donor T cell reconstitution werehighest in animals receiving thymic irradiation or receiving additionalanti-T cell monoclonal antibody treatments following bone marrowtransplantation. Eleven of 20 mice receiving two pre-bone marrowtransplantation monoclonal antibody treatments (the monoclonal antibodytreatments were spaced about 5 days apart with the final treatment 1 dayprior to bone marrow transplantation) and no thymic irradiation showedrelatively low levels of donor T cell reconstitution (less than 20%donor, more than 80% host) at 6 weeks, and 9 of these showed a markedloss of donor cells in all lineages by 20 weeks. In contrast, 12 of 12similarly-treated mice receiving 1 or 2 additional post-bone marrowtransplantation monoclonal antibody treatments (the monoclonal antibodytreatments were spaced about 7 days apart with the first treatment 7 dayafter bone marrow transplantation) showed high levels of donor T cellreconstitution at 6 weeks (mean 86±12% donor), and high levels of donorreconstitution persisted in all lineages at 20 weeks. Thus, a seconddose of pre-bone marrow transplantation T cell-depleting monoclonalantibodies can replace thymic irradiation and allow tolerance inductionin our regimen, but additional monoclonal antibodies administered at oneand two weeks post-bone marrow transplantation may increase the abilityto reliably induce durable mixed chimerism and tolerance. The capacityof repeated anti-T cell monoclonal antibody treatments to replace thymicirradiation in this regimen most likely reflects their ability todeplete host thymocytes that escape depletion by the initial monoclonalantibody treatment. These monoclonal antibodies deplete most host Tcells and induce down-modulation of both TCR and CD4 and CD8co-receptors on the few remaining cells. In these animals, earlymigration of donor bone marrow-derived cells to the host thymus isassociated with complete clonal deletion of mature host-type thymocyteswith TCR that recognize donor antigens. Although a small population ofhost T cells with such TCR persists in the spleens of chimeras, thesecells are anergic to stimulation through their TCR. These cells may haveescaped depletion by down-modulating CD4 or CD8 after monoclonalantibody treatment. Thus, this relatively non-toxic regimen achievespluripotent hematopoietic stem cell engraftment and specific toleranceby ablating most of the existing T cell repertoire and allowing new Tcell development in the presence of intrathymic donor antigen, and byinducing anergy among the few remaining host T cells in the periphery.

Some of the methods herein include the administration of hematopoieticstem cells to a recipient. In many of those methods, hematopoietic stemcells are administered prior to or at the time of the implantation of agraft (an allograft or a xenograft), the primary purpose of theadministration of hematopoietic stem cells being the induction oftolerance to the graft. The inventors have found that one or moresubsequent administrations (e.g., a second, third, fourth, fifth, orfurther subsequent administration) of hematopoietic stem cells can bedesirable in the creation and/or maintenance of tolerance. Thus, theinvention also includes methods in which hematopoietic stem cells areadministered to a recipient, e.g., a primate, e.g., a human, which haspreviously been administered hematopoietic stem cells as part of any ofthe methods referred to herein.

While not wishing to be bound by theory the inventor believes thatrepeated stem cell administration may promote chimerism and possiblylong-term deletional tolerance in graft recipients. Accordingly, anymethod referred to herein which includes the administration ofhematopoietic stem cells can further include multiple administrations ofstem cells. In preferred embodiments: a first and a secondadministration of stem cells are provided prior to the implantation of agraft; a first administration of stem cells is provided prior to theimplantation of a graft and a second administration of stem cells isprovided at the time of implantation of the graft. In other preferredembodiments: a first administration of stem cells is provided prior toor at the time of implantation of a graft and a second administration ofstem cells is provided subsequent to the implantation of a graft. Theperiod between administrations of hematopoietic stem cells can bevaried. In preferred embodiments a subsequent administration ofhematopoietic stem cell is provided: at least two days, one week, onemonth, or six months after the previous administration of stem cells; atleast two days, one week, one month, or six months after theimplantation of the graft.

The method can further include the step of administering a second orsubsequent dose of hematopoietic stem cells: when the recipient beginsto show signs of rejection, e.g., as evidenced by a decline in functionof the grafted organ, by a change in the host donor specific antibodyresponse, or by a change in the host lymphocyte response to donorantigen; when the level of chimerism decreases; when the level ofchimerism falls below a predetermined value; when the level of chimerismreaches or falls below a level where staining with a monoclonal antibodyspecific for a donor PBMC antigen is equal to or falls below stainingwith an isotype control which does not bind to PBMC's, e.g. when thedonor specific monoclonal stains less than 1-2% of the cells; orgenerally, as is needed to maintain tolerance or otherwise prolong theacceptance of a graft. Thus, method of the invention can be modified toinclude a further step of determining if a subject which has received aone or more administrations of hematopoietic stem cells is in need of asubsequent administration of hematopoietic stem cells, and if so,administering a subsequent dose of hematopoietic stem cells to therecipient.

Any of the methods referred to herein can include the administration ofagents, e.g., 15-deoxyspergualin, mycophenolate mofetil, brequinarsodium, or similar agents, which inhibit the production, levels, oractivity of antibodies in the recipient. One or more of these agents canbe administered: prior to the implantation of donor tissue, e.g., one,two, or three days, or one, two, or three weeks before implantation ofdonor tissue; at the time of implantation of donor tissue; or afterimplantation of donor tissue, e.g., one, two, or three days, or one, twoor three weeks after, implantation of a graft.

The administration of the agent can be initiated: when the recipientbegins to show signs of rejection, e.g., as evidenced by a decline infunction of the grafted organ, by a change in the host donor specificantibody response, or by a change in the host lymphocyte response todonor antigen; when the level of chimerism decreases; when the level ofchimerism falls below a predetermined value; when the level of chimerismreaches or falls below a level where staining with a monoclonal antibodyspecific for a donor PBMC antigen is equal to or falls below stainingwith an isotype control which does not bind to PBMC's, e.g. when thedonor specific monoclonal stains less than 1-2% of the cells; orgenerally, as is needed to maintain tolerance or otherwise prolong theacceptance of a graft.

The period over which the agent is administered (or the period overwhich clinically effective levels are maintained in the subject) can belong term, e.g., for six months or more or a year or more, or shortterm, e.g., for less than a year, more preferably six months or less,more preferably one month or less, and more preferably two weeks orless. The period will generally be at least about one week andpreferably at least about two weeks in duration. In preferredembodiments the period is two or three weeks long.

Preferred embodiments include administration of 15-deoxyspergualin (6mg/kg/day) for about two weeks beginning on the day of graftimplantation.

Some of the methods referred to herein include steps in whichantibodies, e.g., preformed natural antibodies, are removed from theblood of a recipient. For example, in some methods antibodies areremoved by hemoperfusion of an organ from the donor species. Theinventor has discovered that an α1-3 galactose linkage epitope-affinitymatrix, e.g., in the form of an affinity column, is useful for removingantibodies from the recipient's blood. Accordingly, the use of an α 1-3galactose linkage epitope-affinity matrix, e.g., matrix bound linear Btype VI carbohydrate, can be added to any method referred to herein andcan be used in addition to or in place of any antibody perfusion orremoval technique, e.g., organ perfusion, in any method referred toherein.

Some of the methods referred to herein include the administration ofhematopoietic stem cells to a recipient. In many of those methodshematopoietic stem cells are administered prior to or at the time of theadministration of a graft (an allograft or a xenograft), the primarypurpose of the administration of hematopoietic stem cells being theinduction of tolerance to the graft. The inventors have found thatadministration of one or more cytokines, preferably a cytokine from thespecies from which the stem cells are derived, can promote tolerance orotherwise prolong acceptance of a graft. Thus, the invention alsoincludes methods in a subject which has previously been administereddonor hematopoietic stem cells, is administered one or more cytokine,e.g., a donor-species cytokine.

Although not wishing to be bound by theory, the inventor believes thatthe cytokines, particularly donor species cytokines, promote theengraftment and/or function of donor stem cells or their progeny cells.Accordingly, any method referred to herein which includes theadministration of hematopoietic stem cells can further includethe-administration of a cytokine, e.g., SCF, IL-3, or GM-CSF. Inpreferred embodiments the cytokine one which is species specific in itsinteraction with target cells.

Administration of a cytokine can begin prior to, at, or after theimplantation of a graft or the implantation of stem cells.

The method can further include the step of administering a first orsubsequent dose of a cytokine to the recipient: when the recipientbegins to show signs of rejection, e.g., as evidenced by a decline infunction of the grafted organ, by a change in the host donor specificantibody response, or by a change in the host lymphocyte response todonor antigen; when the level of chimerism decreases; when the level ofchimerism falls below a predetermined value; when the level of chimerismreaches or falls below a level where staining with a monoclonal antibodyspecific for a donor PBMC antigen is equal to or falls below stainingwith an isotype control which does not bind to PBMC's, e.g. when thedonor specific monoclonal stains less than 1-2% of the cells; orgenerally, as is needed to maintain tolerance or otherwise prolong theacceptance of a graft. Thus, method of the invention can be modified toinclude a further step of determining if a subject is in need ofcytokine therapy and if so, administering a cytokine.

The period over which the cytokine(s) is administered (or the periodover which clinically effective levels are maintained in the subject)can be long term, e.g., for six months of more or a year or more, orshort term, e.g., for a year or less, more preferably six months orless, more preferably one month or less, and more preferably two weeksor less. The period will generally be at least about one week andpreferably at least about two weeks in duration.

In preferred embodiments the recipient is a primate, e.g., a human, andthe donor is from a different species, e.g., the donor is a pig and: pigSCF is administered; pig IL-3 is administered; a combination of pig SCFand pig IL-3 is administered; a pig specific hematopoiesis enhancingfactor, e.g., pig GM-SCF, is administered, e.g., after the implantationof stem cells, e.g., about a month after the implantation of stem cells.

A particularly preferred embodiment combines a short course, e.g., abouta month, of cyclosporine or a similar agent, a short course, e.g., abouttwo weeks, of 15-deoxyspergualin or a similar agent, and a short course,e.g., about two weeks, of donor specific cytokines, e.g., SCF and IL-3.In Cynomolgus monkeys receiving pig grafts and pig stem cells, treatmentwhich included the combination of cyclosporine (15 mg/kg/day for 28days), 15-deoxyspergualin (6 mg/kg/day for two weeks), and recombinantpig cytokines (SCF and IL-3, each at 10 μg/kg/day, i.v., for two weeks)was found to be useful. Administration began at the time of graftimplant. (The monkeys were also given a preparative regimen consistingof 3×100 cGy total body irradiation on day −6, and −5 and hemoperfusionwith a pig liver just prior to stem cell administration.)

An anti-CD2 antibody, preferably a monoclonal, e.g., BTI-322, or amonoclonal directed at a similar or overlapping epitope, can be used inaddition to or in place of any anti-T cell antibodies (e.g., ATG) in anymethod referred to herein.

1. A method of restoring or promoting the thymus-dependent ability for Tcell progenitors to develop into mature functional T cells in a primaterecipient which is capable of producing T cell progenitors but which isthymus-function deficient, the method comprising introducing into saidprimate recipient xenogeneic fetal or neonatal thymus tissue from adonor animal of a different species, so that recipient T cells canmature in said implanted donor thymus tissue.
 2. The method of claim 1,wherein the donor animal is a swine.
 3. The method of claim 2, furthercomprising implanting swine fetal liver tissue in said recipient.
 4. Themethod of claim 2, wherein said primate recipient is a human.
 5. Themethod of claim 2, wherein said donor swine is a miniature swine.
 6. Themethod of claim 2, wherein said swine thymus tissue is capable ofsupporting in the recipient primate clonal deletion or anergy ofthymocytes reactive with donor xenoantigens.
 7. The method of claim 2,further comprising introducing swine hematopoietic cells into saidrecipient and inactivating NK cells of said recipient.
 8. The method ofclaim 2, further comprising the step of inactivating mature CD4⁺ T cellsin the recipient.
 9. A method of inducing tolerance in a recipientprimate of a first species to a graft obtained from a donor animal of asecond discordant species, said method comprising prior or simultaneouswith transplantation of said graft, introducing into said recipient,thymic tissue from the second species; and implanting said graft in saidrecipient.
 10. The method of claim 9, wherein the donor animal is aswine.
 11. The method of claim 10, wherein the same swine is the donorof both the graft and the thymic tissue.
 12. The method of claim 9,wherein said primate is a human.
 13. The method of claim 9, said methodfurther comprising the step of, prior or simultaneous withtransplantation of said graft, inactivating mature CD4⁺ T cells of therecipient.
 14. The method of claim 9, further comprising the step of,prior to thymic tissue transplantation, irradiating the recipient withlow dose whole body irradiation.
 15. The method of claim 14, whereinsaid low dose irradiation is at least 100 rads and less than 400 rads.16. The method of claim 9, further comprising the step, prior to thymictissue transplantation, adsorbing natural antibodies from the blood ofsaid recipient.
 17. The method of claim 9, wherein said thymic tissue iscapable of supporting, in the recipient primate, clonal deletion oranergy of thymocytes reactive with donor xenoantigens.
 18. The method ofclaim 9, wherein hematopoietic cells are administered to said recipientand wherein the method includes inactivating NK cells of said recipient.19. The method of claim 9, wherein said primate recipient is a human andsaid donor is a miniature swine.
 20. A method of providing maturerecipient T cells in a primate recipient which is thymus functiondeficient, comprising introducing into the primate recipient swinexenogeneic donor thymic tissue, so that recipient T cells can mature insaid implanted swine donor thymic tissue.
 21. The method of claim 20,wherein said primate is a human and said donor is a miniature swine. 22.The method of claim 20, wherein said thymus function is deficient insaid recipient due to an immune disorder. 23.-39. (canceled)